Mixed cell diagnostic systems

ABSTRACT

The present invention generally relates to the field of diagnostic microbiology, and, more particularly, to compositions and methods for detecting and differentiating one or more viruses or other intracellular parasites present in a specimen. The present invention also provides compositions and methods to evaluate the susceptibility of a organisms to antimicrobial agents.

[0001] This is a continuation-in-part of copending application Ser. No.09/066,072, filed on Apr. 24, 1998.

FIELD OF THE INVENTION

[0002] The present invention generally relates to the field ofdiagnostic microbiology, and more particularly, to compositions andmethods for detecting and differentiating one or more viruses or otherintracellular parasites present in a specimen. The present inventionalso provides compositions and methods to evaluate the susceptibility ofa organisms to antimicrobial agents.

BACKGROUND OF THE INVENTION

[0003] Despite recent advances in methods for the detection of virusesusing molecular methods, the detection and identification of theseorganisms in cell culture remains the “gold standard” by which mostviral diseases are definitively diagnosed and the method against whichnewer methods are compared (See e.g., Wiedbrauk and Johnston, Manual ofClinical Virology, Raven Press, Inc., New York, N.Y. [1993], pp. 1-17).Cell cultures are also used for the detection and identification ofother intracellular parasites, especially obligate intracellularparasites such as Chlamydia and Rickettsia.

[0004] There are two general types of cell culture methods used forvirus identification. The first method uses identification ofvirus-induced cytopathic effect (CPE) as an endpoint for virusdetection. The second method utilizes molecular methods to identify thepresence of virus before CPE is evident in the infected cultures. Bothmethods utilize cell cultures, which may present problems for smalllaboratories with limited expertise in cell culturing methods, space,funding, equipment, and supplies. Depending upon the cells used, cellcultures can be difficult to maintain and often require the efforts ofskilled laboratorians. In addition, cell cultures require equipment suchas cell culture hoods, inverted microscopes (for observation of cells),incubators with CO₂ lines, and other equipment not readily available inmany laboratories.

[0005] CPE-Based Tests

[0006] CPE-based tests often require long incubation times, asvirus-induced CPE only becomes evident after multiple rounds of viralreplication and spread of virus to neighboring cells (i.e., the cellsare “permissive” for viral infection). Virus spread results in thedestruction of the cells surrounding the cell originally infected.CPE-based tests have been traditionally conducted in tubes or flaskscontaining a single cell type that is adhered or anchored to the sidesand/or bottom of the tube or flask. As the virus must infect a cell,replicate, and spread to neighboring cells in which the process isrepeated, results can be delayed for at least 28 days. Indeed, resultsare often not available for 7-28 days after inoculation of the cellculture with the virus suspension (See e.g., Leland, Clinical Virology,W. B. Saunders, Philadelphia [1996], pp. 60-65). The time necessary toestablish visible CPE is dependent upon the rate of viral replication,which can vary among cell types and viruses. Thus, the amount of timeneeded to detect virus in a sample can greatly vary.

[0007] Pre-CPE Tests

[0008] In contrast to CPE-based tests, pre-CPE tests require only entryof the virus into a susceptible host cell and detectable expression ofat least one early virus-specific antigen or nucleic acid. Detection ofthe virus-specific analyte or other indicator is accomplished by anumber of methods (e.g., labeled antibodies, the polymerase chainreaction [PCR], or the use of other reporters, such as the ELVIS™system). Expression of early viral genes has been shown to be very rapidin many virus-host cell systems in vitro. Thus, use of pre-CPE basedvirus tests significantly reduces the time required to detect andidentify viruses in clinical specimens.

[0009] Pre-CPE detection of virus is often accomplished by usingmonolayers of adherent cells grown on 12 mm round coverslips containedin 1 dram shell vials (i.e., the “shell vial” method or technique). Theshell vial technique uses centrifugation of the specimen to force viralintroduction into cells and enhance viral isolation. These vials areprepared by dispensing cells into sterile shell vials containingcoverslips. The vial are incubated in an upright position until thecells form a monolayer on the coverslip. For shell vial inoculation, theculture medium is decanted from the vial, processed sample (i.e., theclinical specimen) is added to the cell monolayer, and the vial iscentrifuged at low speed, often for one hour. After centrifugation,fresh culture medium is added to each vial. The vials are then incubatedfor the desired period of time. At the end of the incubation period, thecoverslips are stained using an antigen detection method (e.g.,immunofluorescence) or the cells are evaluated via molecular diagnostictechniques.

[0010] In addition to viruses, shell vials are also commonly used forthe detection and identification of Chlamydia, as other methodsavailable for the detection and identification of these organisms arequite cumbersome, as well as time and reagent-consuming (See e.g.,Wiedbrauk and Johnston, supra, pp. 64-76).

[0011] The major advantage of these pre-CPE testing methods is thatrapid test results are often possible. One major disadvantage to pre-CPEtesting of shell vial cultures is that this type of test is feasible andcost-effective only if one or a few viral agents are sought foridentification, and if a high proportion of specimens are likely to bepositive (See e.g., Schmidt and Emmons, “General Principles ofLaboratory Diagnostic Methods for Viral, Rickettsial, and ChlamydialInfections,” in Schmidt and Emmons (eds.), Diagnostic Procedures forViral, Rickettsial and Chlamydial Infections, American Public HealthAssociation, Washington, D.C., [1989], at p. 4).

[0012] Clinical Specimens

[0013] For example, the presence of skin vesicles in the genital area ofa patient is highly suspicious for infection by herpes simplex virus(HSV). Typically, the physician will obtain a specimen from the affectedregion (i.e., a vesicle) and order a CPE or a pre-CPE virus test on asingle, HSV-susceptible cell line. These cell lines are often suppliedeither in tubes, shell vials, or multi-well plates (e.g., microtiterplates). After inoculation of the cell line and an appropriateincubation time, confirmation of the presence of HSV in the sample canbe accomplished using one or more of the many analytical methods (e.g.,immunofluorescence, immunoperoxidase, nucleic acid probes, or substratesfor virus-induced reporter genes).

[0014] For detection of cytomegalovirus (CMV), shell vials containingcells from a single cell line (e.g., human fibroblast cell lines, suchas lung [MRC-5 cells] or foreskin [HFF] cells) are often used. The cellsare grown to confluency on the coverslip within the vial, the sample isadded to the vial, the vial is incubated for 24-48 hours or longer, andan immunofluorescent method is used to detect expression of CMV earlyantigen.

[0015] Accurate differential diagnosis is significantly more difficultin virus diseases due to respiratory, gastrointestinal, genital, orparenteral routes of transmission because many pathogenic viruses arecapable of eliciting similar symptoms or the infection is sub-clinical(i.e., the signs and symptoms are not readily apparent).

[0016] Of the respiratory viruses, rhinoviruses and corona viruses areresponsible for a large proportion of upper respiratory infections. Oncethese viruses reach the upper respiratory mucosa, they attach to andinfect epithelial cells. Typically, these infections last only a fewdays and self-resolve. Other respiratory viruses, such as theinfluenzas, parainfluenzas, respiratory syncytial virus (RSV), andvarious adenoviruses attach to and infect ciliated, columnar epithelialcells. The virus-infected cells lyse, resulting in the release ofenzymes and activate complement, resulting in a local mononuclearinflammatory response. Normal airway clearance mechanisms fail becauseof the failure of the epithelial cells to function normally. These cellsmay also slough off. Cell debris from dead and dying cells oftenobstructs airways, and the host becomes very susceptible to secondarybacterial infection and/or superinfection. All of these viruses mayprogress to lower respiratory involvement and pneumonia. Afterreplication in the respiratory epithelial cells, adenovirus may travelvia the blood to the lymphoid tissues in all areas of the body, causingsystemic infection or disease.

[0017] Standard clinical virology practice is to inoculate multipletubes of cell cultures with the specimen (e.g., throat swab,nasopharyngeal swab, or sputum specimen) as the tropism of each type ofvirus for specific cell types is often very narrow (i.e., only one typeof virus may grow optimally on a single cell type). This narrow tropismof virus for a limited number of cell types creates at least two majorpractical problems for both CPE and pre-CPE virus testing.

[0018] First, primary monkey kidney cells are currently the cell line ofchoice for isolation of influenza viruses. The manufacture of thesecells requires the quarantine of source animals for long periods priorto sacrifice and cell culture preparation. This quarantine period isused to monitor the animals for good health and allows time to test theanimals for infection by endogenous simian viruses such as foamy virus,SV5, and SV40. The quarantine period also greatly reduces, but does noteliminate, the possibility that the monkeys are infected with Monkey BVirus, a herpesvirus that is highly fatal to humans. In addition, thereare other problems related to the use of monkeys for the production ofprimary cell cultures, including the reduction in the stock of suitableanimals due to importation concerns and monkey populationconsiderations.

[0019] Second, additional continuous cell lines are required in order todetect respiratory viruses other than influenza virus. Thus, multiplecell lines are used in order to diagnose the viral infection/disease ofeach patient. The need for multiple units of individual cell lines iscompounded in methods using pre-CPE tests for detection andidentification of respiratory viruses. Pre-CPE testing for respiratoryviruses requires the expenditure of significant labor in handlingcoverslips, the added expense of molecular reagents used with multiplecell lines for both positive and negative specimens, and the significantlabor associated with microscopically reading each of the multiple celllines inoculated in the panel of cell lines.

[0020] However, despite these drawbacks, shell vial technology usingsingle cell types in multiple units (tubes, shell vials, etc.), is stillcurrently used to detect respiratory viruses, as it is a proven method.For example, detection of RSV in 16 hours using shell vials containingonly HEp-2 cells yielded more positives than antigen detection methodsapplied directly to the clinical specimen, and as many positives asconventional cell cultures (Smith et al, J. Clin. Microbiol., 29:463-465[1991]). Isolation of other respiratory viruses has also been possiblewith shell vial cultures containing a monolayer of a single cell type.For example, using vials of primary monkey kidney cells and A549 cellsincubated for 40 hours, 83% of adenoviruses, 94% of influenza B, and 80%of parainfluenza virus types 1, 2, and 3 were identified (Rabalais etal., J. Clin. Microbiol., 30:1505-1508 [1992]). In another report, 50%of adenoviruses, 94% of influenza A viruses, 100% of influenza Bviruses, and 100% of parainfluenza viruses, in shell vials of primaryrhesus monkey kidney cells, and 92% of RSV in shell vials of HEp-2 cellsincubated for 2-4 days (See e.g., Olsen et al., J. Clin. Microbiol.,31:422-425 [1993]; and Leland, Clinical Virology, W. B. SaundersCompany, Philadelphia, Pa. [1996], at p. 85-86).

[0021] Although these methods provide relatively rapid results (i.e., asopposed to the long incubation periods often necessary for CPE tests),there remains a need in clinical and reference virology laboratories forcell culture methods and compositions for the reliable detection andidentification of viruses in a single, easy-to-manipulate unit thatprovides rapid detection and identification in a cost-effective manner,while also providing the sensitivity of a diagnostic assay system.

SUMMARY OF THE INVENTION

[0022] The present invention generally relates to the field ofdiagnostic microbiology, and more particularly, to compositions andmethods for detecting and differentiating one or more viruses or otherintracellular parasites present in a specimen. The present inventionalso provides compositions and methods to evaluate the susceptibility ofa organisms to antimicrobial agents.

[0023] In particular, the present invention provides methods andcompositions suitable for the detection of viruses using CPE-based andpre-CPE methods. The preferred embodiments encompass mixed cell cultureswhich contain at least two different cell types. In some preferredembodiments, the mixed cell cultures contain two different cell types,while in other embodiments, the mixed cell cultures contain three ormore different cell types. Thus, it is intended that the presentinvention encompass compositions in which at least two cell types aremixed together in one container (e.g., flask, tube, shell vial, or anyother container suitable for the growth of cells). Importantly, eachcell type within these mixed cell cultures retains its susceptibility toviruses and other intracellular parasites as if it was in a single cellculture (i.e., a cell culture that contains only one cell type, as knownin the art). In addition, the mixed cell cultures of the presentinvention remain viable for as long as required for their use indiagnostic assays. In particularly preferred embodiments, the cell typesincluded within mixed cell cultures are present in approximately thesame ratio (i.e., for a two cell type mixed, there is a 50:50 ratio ofcell types). However, it is not intended that the present invention belimited to any particular ratio of cell types in mixed culture, asvarious detection systems may be optimized using different ratios. Forexample, in some circumstances, a two cell mixture of 45:55, 40:60, oreven 35:75, may be more suited than a 50:50 ratio.

[0024] The present invention also provides methods and compositionssuitable for the detection and identification of non-viral obligateintracellular and intracellular parasites, such as members of theChlamydiales and Ricketsiales.

[0025] The present invention also contemplates compositions comprising acell culture suitable for the detection of intracellular parasites,wherein the cell culture comprises at least two cell types susceptibleto infection by at least one intracellular parasite. In some preferredembodiments of the composition, the cell types comprise a first celltype and a second cell type. In some embodiments, the first cell typeconsists of buffalo green monkey kidney cells and the second cell typeconsists of mink lung cells. In other embodiments, the first cell typeconsists of mink lung cells and the second cell type consists of humanmucoepidermoid cells. In yet other embodiments, the first cell typeconsists of human lung carcinoma cells and the second cell type consistsof human mucoepidermoid cells. In still other embodiments, the firstcell type consists of buffalo green monkey kidney cells and the secondcell type consists of human embryonic lung cells. In furtherembodiments, the cell type consists of human epidermoid laryngealcarcinoma cells and the second cell type consists of McCoy cells. Inadditional embodiments, the first cell type consists of mink lung cellsand the second cell type consists of human diploid lung cells.

[0026] In some preferred embodiments, the cell types of the compositionare susceptible to respiratory viruses, including but not limited toinfluenza viruses of any type (e.g., Influenza A, Influenza B, andInfluenza C) and/or strain, RSV, cytomegalovirus, parainfluenza viruses,respiratory syncytial virus, rhinoviruses, coronoviruses, andadenoviruses. In yet other embodiments, the cell types of thecomposition are susceptible to enteroviruses, including but not limitedto any type and/or strain of echovirus, poliovirus, and Coxsackie virus(e.g. Coxsackie A and B viruses), and numbered EV strains. In additionto enteroviruses, it is contemplated that the present inventionencompass cell types that are susceptible to picomaviruses such asHepatitis A.

[0027] The present invention also provides methods for the detection andidentification of intracellular parasites in a sample, comprising thesteps of providing a sample suspected of containing one or moreintracellular parasites; and a mixed cell culture comprising at leasttwo cell types; inoculating the mixed cell culture with the sample toproduce an inoculated culture; and observing the inoculated culture forthe presence of the one or more intracellular parasites.

[0028] In some embodiments of the method, the intracellular parasite isa virus. In some particularly preferred embodiments, the virus isselected from the group consisting of cytomegalovirus, influenzaviruses, parainfluenza viruses, respiratory syncytial virus,rhinoviruses, coronoviruses, and adenoviruses. In yet other embodimentsof the methods, the virus is an enterovirus. In other particularlypreferred embodiments, the enterovirus is selected from the groupconsisting of poliovirus, Coxsackie viruses and echoviruses (e.g.,Coxsackie A and B viruses), and numbered EV strains. In addition toenteroviruses, it is contemplated that the present invention encompasscell types that are susceptible to picomaviruses such as Hepatitis A. Instill other preferred embodiments, the virus is a herpes virus. In otherparticularly preferred embodiments, the herpes virus is selected fromthe group consisting of Herpes Simplex Type 1, Herpes Simplex Type 2,Cytomegalovirus, Varicella-Zoster virus, Epstein-Barr virus, HumanHerpes Virus 6, Human Herpes Virus 7, and Human Herpes Virus 8. In yetother preferred embodiments, the intracellular parasite is a member ofthe genus Chlamydia. In still other particularly preferred embodiments,the intracellular parasite is C. trachomatis.

[0029] In some preferred embodiments of the methods, the cell typescomprise a first cell type and a second cell type. In some preferredembodiments, the first cell type is a mink lung cell, and the secondcell type is a human mucoepidermoid cell. In other preferredembodiments, the first cell type is a buffalo green monkey kidney celland the second cell type is a human mucoepidermoid cell. In yet anotheralternative embodiment, the first cell type is a genetically engineeredbaby hamster kidney cell and the second cell type is a mink lung cell.In still other embodiments, the first cell type is a first geneticallyengineered cell type and the second cell type is a second geneticallyengineered cell type.

[0030] It is contemplated that the methods of the present invention willbe used in conjunction with controls of known positivity and negativityfor the virus(es) and/or other intracellular organism of interest.

[0031] The present invention also provides methods for the detection andidentification of intracellular parasites in a sample, comprising thesteps of providing: a sample suspected of containing one or moreintracellular parasites; and a mixed cell culture comprising a firstcell type and a second cell type; inoculating the mixed cell culturewith the sample to produce an inoculated culture; and observing theinoculated culture for the presence of the one or more intracellularparasites.

[0032] In some particularly preferred embodiments, the intracellularparasite is a virus. In some particularly preferred embodiments, thevirus is selected from the group consisting of cytomegalovirus,influenza viruses, parainfluenza viruses, respiratory syncytial virus,rhinoviruses, coronoviruses, and adenoviruses. In yet other embodimentsof the methods, the virus is an enterovirus. In other particularlypreferred embodiments, the enterovirus is selected from the groupconsisting of poliovirus, Coxsackie viruses and echoviruses (e.g.,Coxsackie A and B viruses), and numbered EV strains. In addition toenteroviruses, it is contemplated that the present invention encompasscell types that are susceptible to picornaviruses such as Hepatitis A.In still other preferred embodiments, the virus is a herpes virus. Inother particularly preferred embodiments, the herpes virus is selectedfrom the group consisting of Herpes Simplex Type 1, Herpes Simplex Type2, Cytomegalovirus, Varicella-Zoster virus, Epstein-Barr virus, HumanHerpes Virus 6, Human Herpes Virus 7, and Human Herpes Virus 8. In yetother preferred embodiments, the intracellular parasite is a member ofthe genus Chlamydia. In still other particularly preferred embodiments,the intracellular parasite is C. trachomatis.

[0033] In some preferred embodiments of the methods, the cell typescomprise a first cell type and a second cell type. In some preferredembodiments, the first cell type is a mink lung cell, and the secondcell type is a human mucoepidermoid cell. In other preferredembodiments, the first cell type is a buffalo green monkey kidney celland the second cell type is a human mucoepidermoid cell. In yet anotheralternative embodiment, the first cell type is a genetically engineeredbaby hamster kidney cell and the second cell type is a mink lung cell.In still other embodiments, the first cell type is a first geneticallyengineered cell type and the second cell type is a second geneticallyengineered cell type.

[0034] It is contemplated that the methods of the present invention willbe used in conjunction with controls of known positivity and negativityfor the virus(es) and/or other intracellular organism of interest.

[0035] The present invention further provides methods for the detectionof influenza virus, comprising the steps of providing a sample suspectedof containing influenza virus, and mink lung cells; inoculating the minklung cells with the sample; and detecting the presence of the influenzawithin the mink lung cells. In particularly preferred embodiments, themink lung cells are Mv1Lu cells. In alternative embodiments, theinfluenza virus is selected from the group consisting of Influenza A,Influenza B, and Influenza C.

[0036] It is contemplated that the methods of the present invention willbe used in conjunction with controls of known positivity and negativityfor the virus(es) and/or other intracellular organism of interest.

[0037] In one embodiment, the present invention provides methods for thedetection of infectious virus in a specimen comprising the steps of: a)providing a specimen suspected of containing a virus, a cell linepermissive for infection by the virus, and a genetically engineered cellline containing an oligonucleotide having a sequence comprising apromoter sequence derived from the virus, wherein the promoter sequenceis operably linked to a reporter gene, and wherein the expression of thereporter gene is dependent upon and quantitatively proportional to thepresence of the virus; b) mixing together the permissive cell line andthe genetically engineered cell line to create a mixed cell culture; c)inoculating the mixed cell culture with the specimen under conditionswhich permit the infection of the mixed cell culture by the virus; andd) detecting the expression of the reporter gene and thereby detectingthe presence of virus in the specimen. In one preferred embodiment, themixed cell culture is a mixture consisting of 80-99% of the permissivecell line and 1-20% of the genetically engineered cell line. In otherpreferred embodiments, the mixed cell culture is a mixture consisting ofequal proportions of the cell types used in the mixture.

[0038] In one embodiment of the method, the genetically engineered cellline contains an oligonucleotide having a sequence comprising aherpesvirus inducible promoter operably linked to a reporter geneselected from the group comprising the Escherichia coli lacZ gene and aluciferase gene. In one preferred embodiment of the method, thegenetically engineered cell line is BHKICP10LacZ. In an alternativepreferred embodiment, the genetically engineered cell line isBHKICP6LacZ. However, it is not intended that the reporter gene belimited to the lacZ and luciferase genes. Indeed, it is contemplatedthat any suitable reporter gene known to those in the art will be usefulin the method of the present invention.

[0039] It is also contemplated that various permissive cell lines willbe useful in the method of the present invention. In one embodiment, thepermissive cell line is permissive for infection with herpesvirus. In aparticularly preferred embodiment, the permissive cell line is MRC-5.

[0040] It is contemplated that the method of the present invention willbe used in conjunction with controls of known positivity and negativityfor the virus(es) of interest. Thus, for mixed cultures in whichgenetically engineered cell lines are used, it is contemplated that thepattern of reporter gene expression present in a test sample (e.g., froma clinical specimen) will be compared to the patterns of reporter geneexpression in control samples known to be positive and/or negative forthe virus(es) of interest. It is also contemplated that effectsunrelated to the expression of the reporter gene will be detectable,including but not limited to CPE. These effects, alone and incombination with the reporter gene expression may be used to detect thepresence of viral infection.

[0041] The present invention also provides methods for the typing ofinfectious herpesvirus in specimens, comprising the steps of: a)providing a specimen suspected of containing one or more members of theherpesvirus family, a cell line permissive for infection by one or moremembers of the herpesvirus family, a genetically engineered cell linecontaining an oligonucleotide having a sequence comprising a promotersequence derived from a member of the herpesvirus family wherein thepromoter sequence is operably linked to a reporter gene, and theexpression of the reporter gene is dependent upon and quantitativelyproportional to the presence of herpesvirus and wherein the expressionof the reporter gene varies in a distinguishable manner as a result ofthe presence of different members of the herpesvirus family; b) mixingtogether the permissive cell line and the genetically engineered cellline to create a mixed cell culture; c) inoculating this mixed cellculture with the specimen under conditions which permit the infection ofthe mixed cell culture by members of the herpesvirus family, wherein theinfection results in a distinguishable pattern of expression by thereporter gene; d) detecting the expression of the reporter gene andthereby detecting the presence of one or more members of the herpesvirusfamily in the specimen; and e) identifying the presence of a specificmember of the herpesvirus family based upon the resultingdistinguishable pattern. It is contemplated that this pattern ofexpression will be observable by various assisted and non-assistedmethods, including visual observation by eye, spectrophotometricobservation, etc. It is not intended that the detection ofdistinguishable pattern(s) be limited to any particular method ofdetection.

[0042] In a preferred embodiment of the typing method of the presentinvention, the mixed cell culture is a mixture consisting of 80-99% ofthe permissive cell line and 1-20% of the genetically engineered cellline. In yet other preferred embodiments, the cell types are inapproximate equal proportions in the mixed cell cultures. As with thefirst method described, it is not intended that the present invention belimited to any particular herpesvirus. In one particular embodiment, themember of the herpesvirus family detected and typed using the method ofthe present invention is selected from the group comprising HSV-1,HSV-2, CMV, VZV, EBV, and human herpes viruses such as HHV-6, HHV-7, andHHV-8. It is intended that one or more herpesviruses may be detected andtyped in one specimen. In this manner, co-infection with multipleherpesviruses may be diagnosed. For example, it is contemplated thatmixed infections with HSV-1 and HSV-2 may be detectable and theinfections distinguished using the methods of the present invention.

[0043] In one embodiment of the typing method, the reporter genecomprises E. coli lacZ gene. However, it is not intended that thereporter gene be limited to lacZ. Indeed, it is contemplated that anyreporter gene may be used in this method. In one particularly preferredembodiment, the detection of the reporter gene is accomplished throughthe use of histochemical staining. It is contemplated that one member ofthe herpesvirus family will produce an histochemically pattern ofexpression that is distinguishable from the histochemical patternsproduced by other members of the herpesvirus family. In this manner, itis possible to use the methods of the present invention to distinguishinfection with one herpesvirus from infection with another herpesvirus.

[0044] It is contemplated that the method of the present invention willbe used in conjunction with controls of known positivity and negativityfor the virus(es) of interest. Thus, it is contemplated that the patternof expression present in a test sample (e.g., from a clinical specimen)will be compared to the patterns of expression in control samples knownto be positive and/or negative for the virus(es) of interest. It is alsocontemplated that effects unrelated to the expression of the reportergene will be detectable, including but not limited to CPE. Theseeffects, alone and in combination with reporter gene expression may beused to detect the presence of viral infection, as well as provideinformation to distinguish between viruses.

[0045] In yet another embodiment, the present invention provides acomposition comprising a mixed cell culture, wherein the mixed cellculture comprises the combination of a genetically engineered cell linetransformed with a promoter sequence from a virus, wherein the promotersequence is operably linked to a reporter gene, and wherein expressionof the reporter gene is dependent upon and quantitatively proportionalto the presence of virus, and a non-engineered cell line which ispermissive for virus infection.

[0046] In one embodiment of the composition, the mixed cell culture is amixture consisting of 1-20% of the genetically engineered cell line and80-99% of the permissive cell line. In yet other preferred embodiments,the cell types are in approximate equal proportions in the mixed cellcultures. In one preferred embodiment of the composition, thegenetically engineered cell line component may comprise a promoter for agene that encodes ribonucleotide reductase. In an alternative preferredembodiment, the promoter may comprise genes that encode one or moresubunits of ribonucleotide reductase. In one particularly preferredembodiment, the genetically engineered cell line is BHKICP10LacZ, whilein another particularly preferred embodiment, the genetically engineeredcell line is BHKICP6LacZ. In an alternative embodiment of thecomposition, the genetically engineered cell line comprises an E. colilacZ gene positioned 3′ to a virus inducible promoter. It iscontemplated that this lacZ gene be positioned immediately 3′ to thisvirus-inducible promoter. However, it is not intended that thesesequences will be contiguous. Indeed, it is contemplated only that thereporter and promoter genes are operably linked. Furthermore, it iscontemplated that the composition will comprise promoter sequences fromany virus, including but not limited to members of the herpesvirusfamily. It is also contemplated that the non-engineered cell line bepermissive for infection by any number of viruses, including but notlimited to members of the herpesvirus family.

[0047] In one preferred embodiment, the composition includes agenetically engineered cell line which includes a promoter for a genethat encodes a ribonucleotide reductase large subunit and the virus is amember of the herpesvirus family selected from the group consisting ofHSV-1, HSV-2, CMV, VZV, EBV, HHV-6, HHV-7, and HHV-8. However, it is notintended that the present invention be limited to any particularherpesvirus. In one preferred embodiment, the genetically engineeredcell line component contains an ICP10 promoter and the herpesvirusfamily member is HSV-2, while in another preferred embodiment, thegenetically engineered cell line comprises an ICP6 promoter and theherpesvirus family member is HSV-1.

[0048] It is contemplated that the detection of reporter gene expressionbe accomplished through the use of various methods, including, but notlimited to calorimetric, fluorimetric or luminometric assays or assaysystems. In one preferred embodiment, the reporter gene encodesβ-galactosidase.

[0049] In one embodiment, the composition includes a geneticallyengineered cell line that is a mammalian cell line susceptible toinfection by virus. In one preferred embodiment, the geneticallyengineered cell line comprises baby hamster kidney cells (e.g., celllines derived from BHK cells). In one embodiment, the compositionincludes a permissive cell line that is permissive to infection byherpesviruses, including but not limited to HSV-1 and HSV-2. In aparticularly preferred embodiment, the permissive cell line is MRC-5. Itis not intended that the composition of the present invention be limitedto detection of viral infection based on the expression of the reportergene, as effects such as CPE may also be detectable.

[0050] The present invention also provides a kit for assaying for thepresence of infectious herpesvirus in a specimen. The kit includes: a) asupply of a mixed cell line comprised of a cell line of geneticallyengineered mammalian cells susceptible to infection by herpesvirus,wherein the cell line contains an oligonucleotide having a sequencecomprising a virus promoter sequence operably linked to a reporter gene,and where the expression of the reporter gene is dependent upon andquantitatively proportional to the presence of virus in the specimen;and a cell line permissive for virus; and b) a supply of reagents todetect the expression of the reporter gene. It is not intended that thepromoter sequences present within the genetically engineered cell linebe limited to any particular virus or virus family. It is contemplatedthat any virus promoter will be useful in the kit of the presentinvention. However, in one preferred embodiment, herpesvirus promotersequences are present in the genetically engineered cell line.

[0051] It is contemplated that various promoter sequences will be usefulin the kit of the present invention. However, in a preferred embodiment,the promoter encodes either a complete ribonucleotide reductase enzyme,or in the alternative, subunits of ribonucleotide reductase. In oneparticularly preferred embodiment, the promoter sequence contains apromoter for a gene that encodes a ribonucleotide reductase largesubunit and the herpesvirus is a herpesvirus family member selected fromthe group consisting of HSV-1, HSV-2, CMV, VZV, EBV, HHV-6, HHV-7, andHHV-8. However, it is not intended that the kit will be limited to thislist of herpesviruses. Indeed, it is contemplated that any herpesvirusmay be detected using the present kit. In one particularly preferredembodiment of the kit, the promoter sequence contains an ICP10 promoterand the herpesvirus family member is HSV-2, while in an alternativepreferred embodiment, the promoter sequence contains an ICP6 promoterand the herpesvirus family member is HSV-1.

[0052] In one preferred embodiment of the kit, the promoter sequencepresent in the genetically engineered cell line comprises an E. colilacZ gene that is operably linked to a herpesvirus inducible promoter.In one particularly preferred embodiment, the genetically engineeredmammalian cells are BHKICP10LacZ cells, while in an alternativeembodiment, the cells are BHKICP6LacZ cells.

[0053] In one preferred embodiment, the reporter gene encodesβ-galactosidase. However, it is not intended that the present inventionbe limited to any particular reporter gene. It is also contemplated thatthe reporter gene will encode any number of enzymes that are amenable todetection by various methods, including but not limited to such methodsas colorimetric, fluorimetric or luminometric assay systems. In onepreferred embodiment of the kit, the reagents provided for the detectionof reporter gene expression may include, but are not limited to,solutions of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside,o-nitrophenyl-galactopyranoside solution, and fluoresceindi-β-D-galactopyranoside. However, it is not intended to limit the kitto these assay systems, as other systems (e.g., radiometric assaysystems) may be useful.

[0054] It is contemplated that the kit of the present invention may alsoinclude additional components, such as materials suitable for positiveand negative controls and instructions for use. It is not intended thatthe kit of the present invention be limited to the mixed cell line andreagents for the detection of reporter gene expression. It is alsointended that the kit will be useful for detection of viral effects oncells other than and unrelated to reporter gene expression. For example,it is contemplated that the kit may be useful for detection of CPE.

[0055] The invention further provides methods for the detection andidentification of enterovirus in a sample by using mixed cell typecultures which contain RD cells and human mucoepidermoid cells. Inparticular, the invention provides a method for the detection andidentification of enterovirus in a sample, comprising: a) providing: i)a sample suspected of containing enterovirus; and ii) a mixed cellculture comprising at least two cell types, wherein the cell typescomprise RD cells and human mucoepidermoid cells; b) inoculating themixed cell culture with at least a portion of the sample to produce aninoculated culture; and c) observing the inoculated culture for thepresence of the enterovirus. Without intending to limit the invention toany particular mucoepidermoid cell, in one preferred embodiment, thehuman mucoepidermoid cell is H292. Also while not intending to limit theinvention to the type of enterovirus, in an alternative preferredembodiment, the enterovirus is selected from Coxsackie viruses,echoviruses, and polioviruses.

[0056] Also provided herein are methods for the detection andidentification of enterovirus in a sample by using mixed cell typecultures containing Caco-2 cells in combination with other cell types.In particular, the invention provides a method for the detection andidentification of enterovirus in a sample, comprising: a) providing: i)a sample suspected of containing enterovirus; and ii) a mixed cellculture comprising at least two cell types, wherein one of the celltypes comprises Caco-2 cells; b) inoculating the mixed cell culture withat least a portion of the sample to produce an inoculated culture; andc) observing the inoculated culture for the presence of the enterovirus.It is not intended that the invention be limited to the type or sourceof the cell types which are preset in mixed cell culture with Caco-2cells. Nonetheless, in one embodiment, the cell type other than theCaco-2 cells is selected from RD cells, H292 cells, BGMK cells, and A549cells. The methods provided herein are not intended to be limited to thetype of enterovirus. However, in one embodiment, the enterovirus isselected from Coxsackie viruses, echoviruses, polioviruses, rotaviruses,astroviruses, and adenoviruses.

[0057] Also provided by the instant invention are methods for thedetection and identification of parainfluenza virus using mixed celltype cultures which contain mink lung cells. Specifically provided is amethod for the detection and identification of parainfluenza virus in asample, comprising the steps of: a) providing: i) a sample suspected ofcontaining parainfluenza virus; and ii) a mixed cell culture comprisingat least two cell types, wherein one of the cell types comprises minklung cells; b) inoculating the mixed cell culture with at least aportion of the sample to produce an inoculated culture; and c) observingthe inoculated culture for the presence of the parainfluenza virus.Without intending to limit the invention to any particular type of minklung cell, in one preferred embodiment, the mink lung cells are Mv1Lucells. Also without intending to limit the invention to a particularparainfluenza virus, in a preferred embodiment of the methods, theparainfluenza virus is selected from type 1, type 2 and type 3.

[0058] The invention further provides a method for the detection andidentification of cytomegalovirus in a sample, comprising the steps of:a) providing: i) a sample suspected of containing cytomegalovirus; andii) a mixed cell culture comprising MRC-5 cells and at least one celltype selected from McCoy cell, genetically engineered baby hamsterkidney cell, A549 cell, HEp-2 cell, Mv1Lu cell, and H292 cell; b)inoculating the mixed cell culture with the sample to produce aninoculated culture; and c) observing the inoculated culture for thepresence of the cytomegalovirus.

[0059] Also provided herein is a method for the detection andidentification of enterovirus in a sample, comprising the steps of: a)providing: i) a sample suspected of containing enterovirus; and ii) amixed cell culture comprising CV-1 cells and at least one cell typewhich comprises a first cell type selected from MRC-5 cell, McCoy cell,genetically engineered baby hamster kidney cell, A549 cell, HEp-2 cell,Mv1Lu cell, and H292 cell; b) inoculating the mixed cell culture withthe sample to produce an inoculated culture; and c) observing theinoculated culture for the presence of the enterovirus.

[0060] The invention additionally discloses a method for the detectionand identification of enterovirus in a sample, comprising the steps of:a) providing: i) a sample suspected of containing enterovirus; and ii) amixed cell culture comprising buffalo green monkey cells and at leastone cell type which comprises a first cell type selected from MRC-5cell, CV-1 cell, McCoy cell, genetically engineered baby hamster kidneycell, A-549 cell, HEp-2 cell, Mv1Lu cell, and H292 cell; b) inoculatingthe mixed cell culture with the sample to produce an inoculated culture;and c) observing the inoculated culture for the presence of theenterovirus.

[0061] Further provided by this disclosure is a method for the detectionand identification of herpes simplex virus in a sample, comprising thesteps of: a) providing: i) a sample suspected of containing herpessimplex virus; and ii) a mixed cell culture comprising geneticallyengineered baby hamster kidney cells and at least one cell type whichcomprises a first cell type selected from MRC-5 cell, CV-1 cell, buffalogreen monkey kidney cell, McCoy cell, A549 cell, HEp-2 cell, Mv1Lu cell,and H292 cell; b) inoculating the mixed cell culture with the sample toproduce an inoculated culture; and c) observing the inoculated culturefor the presence of the herpes simplex virus.

[0062] Yet another method is provided for the detection andidentification of adenovirus in a sample, comprising the steps of: a)providing: i) a sample suspected of containing adenovirus; and ii) amixed cell culture comprising A549 cells and at least one cell typewhich comprises a first cell type selected from MRC-5 cell, CV-1 cell,buffalo green monkey kidney cell, McCoy cell, genetically engineeredbaby hamster kidney cell, HEp-2 cell, Mv1Lu cell, and H292 cell; b)inoculating the mixed cell culture with the sample to produce aninoculated culture; and c) observing the inoculated culture for thepresence of the adenovirus.

[0063] Also provided herein is a method for the detection andidentification of respiratory syncytial virus in a sample, comprisingthe steps of: a) providing: i) a sample suspected of containingrespiratory syncytial virus; and ii) a mixed cell culture comprisingHEp-2 cells and at least one cell type which comprises a first cell typeselected from MC-5 cell, CV-1 cell, buffalo green monkey kidney cell,McCoy cell, genetically engineered baby hamster kidney cell, A549 cell,Mv1Lu cell, and H292 cell; b) inoculating the mixed cell culture withthe sample to produce an inoculated culture; and c) observing theinoculated culture for the presence of the respiratory syncytial virus.

[0064] Additionally provided is a method for the detection andidentification of a virus selected from influenza virus andparainfluenza virus in a sample, comprising the steps of: a) providing:i) a sample suspected of containing a virus selected from influenzavirus and parainfluenza virus; and ii) a mixed cell culture comprisingMv1Lu cells and at least one cell type which comprises a first cell typeselected from MRC-5 cell, CV-1 cell, buffalo green monkey kidney cell,McCoy cell, genetically engineered baby hamster kidney cell, A549 cell,HEp-2 cell, and H292 cell; b) inoculating the mixed cell culture withthe sample to produce an inoculated culture; and c) observing theinoculated culture for the presence of the virus selected from influenzavirus and parainfluenza virus.

[0065] The invention also provides a method for the detection andidentification of a virus selected from respiratory syncytial virus,adenovirus, cytomegalovirus, and parainfluenza virus in a sample,comprising the steps of: a) providing: i) a sample suspected ofcontaining a virus selected from respiratory syncytial virus,adenovirus, cytomegalovirus, and parainfluenza virus; and ii) a mixedcell culture comprising H292 cells and at least one cell type whichcomprises a first cell type selected from MRC-5 cell, CV-1 cell, buffalogreen monkey kidney cell, McCoy cell, genetically engineered babyhamster kidney cell, A549 cell, HEp-2 cell, and Mv1Lu cell, and a secondcell type; b) inoculating the mixed cell culture with the sample toproduce an inoculated culture; and c) observing the inoculated culturefor the presence of the virus selected from respiratory syncytial virus,adenovirus, cytomegalovirus, and parainfluenza virus. Without intendingto limit the second cell type to any particular cell, in one embodiment,the second cell type is selected from MRC-5 cell, CV-1 cell, buffalogreen monkey kidney cell, genetically engineered baby hamster kidneycell, A549 cell, HEp-2 cell, Mv1Lu cell, and H292 cell.

DESCRIPTION OF THE INVENTION

[0066] The present invention generally relates to the field ofdiagnostic microbiology, and more particularly, to compositions andmethods for detecting and differentiating one or more viruses or otherintracellular parasites present in a specimen. The present inventionalso provides compositions and methods to evaluate the susceptibility ofa organisms to antimicrobial agents.

[0067] The present invention provides methods and compositions for thedetection of several different viruses, as well as other intracellularorganisms present in clinical and other specimens in a single cellculture unit comprised of a mixture of cells grown in a manner toco-exist as a monolayer of relatively equivalent ratio and demonstratingcomplementary susceptibilities to a wider range of viruses and/or otherorganisms than could be detected by each individual cell line. Forexample, the viral assays involve inoculating a cell mixture with aspecimen suspected of containing a virus, allowing a sufficient periodof time for the virus infectious cycle to proceed, followed by thedetection and/or quantification of the number of virus-infected cells todetermine the number of infectious virions in the specimen. Thisdetection step may be accomplished using any number of availableconfirmation methods, including specific viral antigen detection usingantigen-specific antibodies, nucleic acid probes, and reporter genedetection. The assay also provides reliable methods and compositions forthe quantification of the number of infectious virions present in asample. In addition, the methods and compositions of the presentinvention are sufficiently sensitive that the presence of a singlevirion in a specimen may be detected.

[0068] The present invention also provides compositions comprising novelmixtures of various cell types traditionally used in single cell assays.In preferred embodiments, the cells are mixed to produce mixed monolayercell cultures. One such mixed cell culture includes mink lung (e.g.,Mv1Lu) cells co-cultivated with human mucoepidermoid cells (e.g.,NCI-H292; also referred to as “H292” cells). This cell mixture issusceptible to viruses such as influenza A, influenza B, RSV,parainfluenza types 1, 2, and 3, adenovirus, and CMV (i.e., the group ofviruses most commonly associated with respiratory virus disease). Inother mixed cultures, buffalo green monkey kidney cells (BGMK) areco-cultivated with NCI-H292 cells for the detection and identificationof enteroviruses, such as poliovirus, echoviruses and Coxsackie B virus(e.g., Coxsackie A and B viruses), and numbered EV strains. In additionto enteroviruses, it is contemplated that the present inventionencompass cell types that are susceptible to picomaviruses such asHepatitis A.

[0069] The present invention also provides compositions comprising novelmixtures of different cell types traditionally used in single cellassays that are co-cultivated with genetically engineered cells. Inparticularly preferred embodiments, the genetically engineered cell lineis a DNA-transfected cell line that is susceptible to infection by avirus, the cell line having been stably transformed with a chimeric genecomprising a virus-inducible promoter and a gene coding for an enzyme,the expression of the enzyme being dependent upon the presence of thevirus. Such genetically engineered cells are, for example, described inU.S. Pat. No. 5,418,132, herein incorporated by reference. In onepreferred embodiment, a cell mixture includes human lung fibroblasts(e.g., MRC-5 cells) co-cultivated with a stable baby hamster kidney(BHK) cell line, the genome of which has been engineered to contain theE. coli lacZ gene behind (i.e., 3′ to) an inducible HSV promoter, HSV-1ICP6 promoter (BHK/ICP6LacZ-5 cells are available from the ATCC asCRL-12072). This cell mixture is susceptible to infection by CMV and HSVtypes 1 and 2.

[0070] In yet another embodiment, the present invention providescompositions comprising novel mixtures of different types of geneticallyengineered cells. In particularly preferred embodiments, the geneticallyengineered cell line is a DNA-transfected cell line that is susceptibleto infection by a virus, the cell line having been stably transformedwith a chimeric gene comprising a virus-inducible promoter and a genecoding for an enzyme, the expression of the enzyme being dependent uponthe presence of the virus. The second genetically engineered cell lineis a DNA-transfected cell line susceptible to viral infection, which isstably transformed with a chimeric gene comprising a virus-induciblepromoter, and a gene encoding a second enzyme (i.e., an enzyme that isdifferent from that associated with the first cell line), in which theexpression of the second enzyme is dependent upon the presence of asecond virus. In one preferred embodiment, a cell mixture is prepared inwhich engineered BHK cells (e.g., BHK/ICP6LacZ-5 cells) areco-cultivated with a stable mink lung cell line (Mv1Lu), the genome ofwhich has been engineered to contain the an inducible CMV promoter (theCMV UL45 promoter); these cells are referred to as “MILD5” cells, andare disclosed in U.S. Pat. Appln. Ser. No. 08/846,026, hereinincorporated by reference. This cell mixture is susceptible to infectionby CMV and HSV virus types 1 and 2 (HSV-1 and HSV-2), with CMV infectingthe genetically engineered BHK cells, and HSV-1 and HSV-2 preferentiallyinfecting the mink lung cells. In another embodiment, the presentinvention contemplates the use of genetically engineered cells (e.g.,mink lung cells) in which the cell genome is engineered to contain thefirefly luciferase gene behind (i.e., 3′ to) an inducible CMV promoter;these cells are also described in U.S. Pat. Appln. Ser. No. 08/846,026.However, it is not intended that the present invention be limited to anyparticular cell types or cell lines, nor is it intended that the presentinvention be limited to any particular combinations of cells. It is alsonot intended that the present invention be limited in terms of thegenetically engineered cells.

[0071] The following Table provides a matrix which indicates the abilityof various cells to form single, confluent monolayers, as well asco-cultivated, confluent, mixed cell monolayers. TABLE 1 Cell CulturesNCI- MRC-5 CV-1 BGMK McCoy BHK* A549 HEp-2 Mv1Lu H292 1 2 3 4 5 6 7 8 9MRC-5 A ++ + No + + + + + + CV-1 B ++ No + + + + + + BGMK C++ + + + + + + McCoy D ++ + + Yes + + BHK* E ++ + + + + A549 F ++ + + +HEp-2 G ++ + + Mv1Lu H ++ + NCI- I ++ H292

[0072] In yet another embodiment, the present invention provides kitsfor assaying samples for the presence of infectious viruses. In thesekits, mixed cell cultures are provided which facilitate the detectionand identification of particular virus groups (e.g.,viruses associatedwith respiratory infections/diseases). In the kits, co-cultivated cellsare supplied either frozen or dispensed (i.e., ready for use) in shellvials, tubes, or multiwell plates. These cells are susceptible toinfection by the virus group of interest as indicated by the sampletype. In preferred embodiments, the kits also include reagents necessaryto detect expression of viral antigens or virus-induced reporter geneexpression.

[0073] One of the several advantages of the present invention is that itprovides rapid and sensitive assay systems for the detection andidentification of a single virus type from a multiplicity ofpossibilities, in a single mixed cell unit that is suitable fordiagnostic assay. Thus, the present invention eliminates the need formultiple cell lines cultured in individual containers, provides reliableresults in 1-3 days following inoculation of the cell cultures, ratherthan 1-28 days, eliminates the necessity of working with primary cellcultures, provides an efficient screening method for grouping andpreliminary identification of viruses, and provides assay systems thatare highly specific for viruses capable of inducing reporter geneexpression. Thus, the present invention clearly fulfills a need that hasbeen heretofore unmet in the field of diagnostic virology.

[0074] Definitions

[0075] The terms “sample” and “specimen” in the present specificationand claims are used in their broadest sense. On the one hand, they aremeant to include a specimen or culture. On the other hand, they aremeant to include both biological and environmental samples. These termsencompasses all types of samples obtained from humans and other animals,including but not limited to, body fluids such as urine, blood, fecalmatter, cerebrospinal fluid (CSF), semen, sputum, and saliva, as well assolid tissue. These terms also refers to swabs and other samplingdevices which are commonly used to obtain samples for culture ofmicroorganisms.

[0076] Biological samples may be animal, including human, fluid ortissue, food products and ingredients such as dairy items, vegetables,meat and meat by-products, and waste. Environmental samples includeenvironmental material such as surface matter, soil, water, andindustrial samples, as well as samples obtained from food and dairyprocessing instruments, apparatus, equipment, disposable, andnon-disposable items. These examples are not to be construed as limitingthe sample types applicable to the present invention.

[0077] Whether biological or environmental, a sample suspected ofcontaining microorganisms may (or may not) first be subjected to anenrichment means to create a “pure culture” of microorganisms. By“enrichment means” or “enrichment treatment,” the present inventioncontemplates (i) conventional techniques for isolating a particularmicroorganism of interest away from other microorganisms by means of anyculture medium and/or technique, and (ii) novel techniques for isolatingparticular microorganisms away from other microorganisms. It is notintended that the present invention be limited only to one enrichmentstep or type of enrichment means. For example, it is within the scope ofthe present invention, following subjecting a sample to a conventionalenrichment means, to subject the resultant preparation to furtherpurification such that a pure culture of a strain of a species ofinterest is produced. This pure culture may then be analyzed by themedium and method of the present invention.

[0078] As used herein, the term “organism” and “microorganism,” are usedto refer to any species or type of microorganism, including but notlimited to viruses and bacteria, including rickettsia and chlamydia.Thus, the term encompasses, but is not limited to DNA and RNA viruses,as well as organisms within the orders Rickettsiales and Chlamydiales.

[0079] As used herein, the term “culture,” refers to any sample orspecimen which is suspected of containing one or more microorganisms.“Pure cultures” are cultures in which the organisms present are only ofone strain of a particular genus and species. This is in contrast to“mixed cultures,” which are cultures in which more than one genus and/orspecies of microorganism are present.

[0080] As used herein, the term “cell type,” refers to any cell,regardless of its source or characteristics.

[0081] As used herein, the term “cell line,” refers to cells that arecultured in vitro, including primary cell lines, finite cell lines,continuous cell lines, and transformed cell lines.

[0082] As used herein, the terms “primary cell culture,” and “primaryculture,” refer to cell cultures that have been directly obtained fromanimal or insect tissue. These cultures may be derived from adults aswell as fetal tissue.

[0083] As used herein, the term “finite cell lines,” refer to cellcultures that are capable of a limited number of population doublingsprior to senescence.

[0084] As used herein, the term “continuous cell lines,” refer to cellcultures that have undergone a “crisis” phase during which a populationof cells in a primary or finite cell line apparently ceases to grow, butyet a population of cells emerges with the general characteristics of areduced cell size, higher growth rate, higher cloning efficiency,increased tumorigenicity, and a variable chromosomal complement. Thesecells often result from spontaneous transformation in vitro. These cellshave an indefinite lifespan.

[0085] As used herein, the term “transformed cell lines,” refers to cellcultures that have been transformed into continuous cell lines with thecharacteristics as described above. Transformed cell lines can bederived directly from tumor tissue and also by in vitro transformationof cells with whole virus (e.g., SV40 or EBV), or DNA fragments derivedfrom a transforming virus using vector systems.

[0086] As used herein, the term “hybridomas,” refers to cells producedby fusing two cell types together. Commonly used hybridomas includethose created by the fusion of antibody-secreting B cells from animmunized animal, with a malignant myeloma cell line capable ofindefinite growth in vitro. These cells are cloned and used to preparemonoclonal antibodies.

[0087] As used herein, the term “mixed cell culture,” refers to amixture of two types of cells. In some preferred embodiments, the cellsare cell lines that are not genetically engineered, while in otherpreferred embodiments the cells are genetically engineered cell lines.In some embodiments, the one or more of the cell types is re“permissive” (i.e., virus is capable of replication and spread from cellto cell within the culture). The present invention encompasses anycombination of cell types suitable for the detection, identification,and/or quantitation of viruses in samples, including mixed cell culturesin which all of the cell types used are not genetically engineered,mixtures in which one or more of the cell types are geneticallyengineered and the remaining cell types are not genetically engineered,and mixtures in which all of the cell types are genetically engineered.

[0088] As used herein, the term “suitable for the detection ofintracellular parasites,” refers to cell cultures that can besuccessfully used to detect the presence of an intracellular parasite ina sample. In preferred embodiments, the cell cultures are capable ofmaintaining their susceptibility to infection and/or support replicationof the intracellular parasite. It is not intended that the presentinvention be limited to a particular cell type or intracellularparasite.

[0089] As used herein, the term “susceptible to infection” refers to theability of a cell to become infected with virus or another intracellularorganism. Although it encompasses “permissive” infections, it is notintended that the term be so limited, as it is intended that the termencompass circumstances in which a cell is infected, but the organismdoes not necessarily replicate and/or spread from the infected cell toother cells. The phrase “viral proliferation,” as used herein describesthe spread or passage of infectious virus from a permissive cell type toadditional cells of either a permissive or susceptible character.

[0090] As used herein, the terms “monolayer,” “monolayer culture,” and“monolayer cell culture,” refer to cells that have adhered to asubstrate and grow in as a layer that is one cell in thickness.Monolayers may be grown in any format, including but not limited toflasks, tubes, coverslips (e.g., shell vials), roller bottles, etc.Cells may also be grown attached to microcarriers, including but notlimited to beads.

[0091] As used herein, the term “suspension,” and “suspension culture,”refers to cells that survive and proliferate without being attached to asubstrate. Suspension cultures are typically produced usinghematopoietic cells, transformed cell lines, and cells from malignanttumors.

[0092] As used herein, the terms “culture media,” and “cell culturemedia,” refers to media that are suitable to support the growth of cellsin vitro (i.e., cell cultures). It is not intended that the term belimited to any particular culture medium. For example, it is intendedthat the definition encompass outgrowth as well as maintenance media.Indeed, it is intended that the term encompass any culture mediumsuitable for the growth of the cell cultures of interest.

[0093] As used herein, the term “obligate intracellular parasite,” (or“obligate intracellular organism) refers to any organism which requiresan intracellular environment for its survival and/or replication.Obligate intracellular parasites include viruses, as well as many otherorganisms, including certain bacteria (e.g., most members of the ordersRickettsiales [e.g., Coxiella, Rickettsia and Ehrlichia] andChlamydiales [e.g., C. trachomatis, C. psittaci], etc). The term“intracellular parasite,” refers to any organism that may be foundwithin the cells of a host animal, including but not limited to obligateintracellular parasites briefly described above. For example,intracellular parasites include organisms such as Brucella, Listeria,Mycobacterium (e.g., M. tuberculosis and M. leprae), and Plasmodium, aswell as Rochalimea.

[0094] As used herein, the term “antimicrobial,” is used in reference toany compound which inhibits the growth of, or kills microorganisms. Itis intended that the term be used in its broadest sense, and includes,but is not limited to compounds such as antibiotics which are producednaturally or synthetically. It is also intended that the term includescompounds and elements that are useful for inhibiting the growth of, orkilling microorganisms.

[0095] As used herein, the terms “chromogenic compound,” and“chromogenic substrate,” refer to any compound useful in detectionsystems by their light absorption or emission characteristics. The termis intended to encompass any enzymatic cleavage products, soluble, aswell as insoluble, which are detectable either visually or with opticalmachinery. Included within the designation “chromogenic” are allenzymatic substrates which produce an end product which is detectable asa color change. This includes, but is not limited to any color, as usedin the traditional sense of “colors,” such as indigo, blue, red, yellow,green, orange, brown, etc., as well as fluorochromic or fluorogeniccompounds, which produce colors detectable with fluorescence (e.g., theyellow-green of fluorescein, the red of rhodamine, etc.). It is intendedthat such other indicators as dyes (e.g., pH) and luminogenic compoundsbe encompassed within this definition.

[0096] As used herein, the commonly used meaning of the terms “pHindicator,” “redox indicator,” and “oxidation-reduction indicator,” areintended. Thus, “pH indicator,” encompasses all compounds commonly usedfor detection of pH changes, including, but not limited to phenol red,neutral red, bromthymol blue, bromcresol purple, bromcresol green,bromchlorophenol blue, m-cresol purple, thymol blue, bromcresol purple,xylenol blue, methyl red, methyl orange, and cresol red. The terms“redox indicator,” and “oxidation-reduction indicator,” encompasses allcompounds commonly used for detection of oxidation/reduction potentials(i.e., “eH”) including, but not limited to various types or forms oftetrazolium, resazurin, and methylene blue.

[0097] As used herein, the term “inoculating suspension,” or“inoculant,” is used in reference to a suspension which may beinoculated with organisms to be tested. It is not intended that the term“inoculating suspension,” be limited to a particular fluid or liquidsubstance. For example, inoculating suspensions may be comprised ofwater, saline, or an aqueous solution. It is also contemplated that aninoculating suspension may include a component to which water, saline orany aqueous material is added. It is contemplated in one embodiment,that the component comprises at least one component useful for theintended microorganism. It is not intended that the present invention belimited to a particular component.

[0098] As used herein, the term “kit,” is used in reference to acombination of reagents and other materials.

[0099] As used herein, the term “primary isolation,” refers to theprocess of culturing organisms directly from a sample. As used herein,the term “isolation,” refers to any cultivation of organisms, whether itbe primary isolation or any subsequent cultivation, including “passage,”or “transfer,” of stock cultures of organisms for maintenance and/oruse.

[0100] As used herein, the term “presumptive diagnosis,” refers to apreliminary diagnosis which gives some guidance to the treatingphysician as to the etiologic organism involved in the patient'sdisease. Presumptive diagnoses are often based on “presumptiveidentifications,” which as used herein refer to the preliminaryidentification of a microorganism.

[0101] As used herein, the term “definitive diagnosis,” is used to referto a final diagnosis in which the etiologic agent of the patient'sdisease has been identified. The term “definitive identification” isused in reference to the final identification of an organism to thegenus and/or species level.

[0102] The term “recombinant DNA molecule,” as used herein refers to aDNA molecule which is comprised of segments of DNA joined together bymeans of molecular biological techniques.

[0103] DNA molecules are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. Therefore, an end of an oligonucleotides is referred to as the“5′ end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. In either alinear or circular DNA molecule, discrete elements are referred to asbeing “upstream” or 5′ of the “downstream” or 3′ elements. Thisterminology reflects the fact that transcription proceeds in a 5′ to 3′fashion along the DNA strand. The promoter and enhancer elements whichdirect transcription of a linked gene are generally located 5′ orupstream of the coding region (enhancer elements can exert their effecteven when located 3′ of the promoter element and the coding region).Transcription termination and polyadenylation signals are located 3′ ordownstream of the coding region.

[0104] The term “an oligonucleotide having a nucleotide sequenceencoding a gene,” refers to a DNA sequence comprising the coding regionof a gene or, in other words, the DNA sequence which encodes a geneproduct. The coding region may be present in either a cDNA or genomicDNA form. Suitable control elements such as enhancers, promoters, splicejunctions, polyadenylation signals, etc. may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Alternatively, the coding region utilized in the vectors ofthe present invention may contain endogenous enhancers and/or promoters,splice junctions, intervening sequences, polyadenylation signals, etc.or a combination of both endogenous and exogenous control elements.

[0105] The term “transcription unit,” as used herein refers to thesegment of DNA between the sites of initiation and termination oftranscription and the regulatory elements necessary for the efficientinitiation and termination. For example, a segment of DNA comprising anenhancer/promoter, a coding region, and a termination andpolyadenylation sequence comprises a transcription unit.

[0106] The term “regulatory element,” as used herein refers to a geneticelement which controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element whichfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements are splicing signals, polyadenylationsignals, termination signals, etc. (defined infra).

[0107] The terms “reporter gene construct,” or “reporter gene vector,”as used herein refers to a recombinant DNA molecule containing asequence encoding the product of a reporter gene and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Eukaryotic cells areknown to utilize promoters, enhancers, and termination andpolyadenylation signals.

[0108] The term “reporter gene,” refers to an oligonucleotide having asequence encoding a gene product (typically an enzyme) which is easilyand quantifiably assayed when a construct comprising the reporter genesequence operably linked to a heterologous promoter and/or enhancerelement is introduced into cells containing (or which can be made tocontain) the factors necessary for the activation of the promoter and/orenhancer elements. Examples of reporter genes include but are notlimited to bacterial genes encoding β-galactosidase (lacZ), thebacterial chloramphenicol acetyltransferase (cat) genes, fireflyluciferase genes and genes encoding β-glucuronidase (GUS).

[0109] Transcriptional control signals in eukaryotes comprise “promoter”and “enhancer” elements. Promoters and enhancers consist of short arraysof DNA sequences that interact specifically with cellular proteinsinvolved in transcription (Maniatis, et al., Science 236:1237 [1987]).Promoter and enhancer elements have been isolated from a variety ofeukaryotic sources including genes in yeast, insect and mammalian cellsand viruses (analogous control elements [i.e., promoters, are also foundin prokaryotes]). The selection of a particular promoter and enhancerdepends on what cell type is to be used to express the protein ofinterest. Some eukaryotic promoters and enhancers have a broad hostrange while others are functional in a limited subset of cell types (forreview see Voss, et al., Trends Biochem. Sci., 11:287 [1986], andManiatis, et al., supra [1987]). For example, the SV40 early geneenhancer is very active in a wide variety of cell types from manymammalian species and has been widely used for the expression ofproteins in mammalian cells (Dijkema, et al., EMBO J. 4:761 [1985]). Twoother examples of promoter/enhancer elements active in a broad range ofmammalian cell types are those from the human elongation factor 1α gene(Uetsuki et aL, J. Biol. Chem., 264:5791 [1989]; Kim et al., Gene 91:217[1990]; and Mizushima and Nagata, Nuc. Acids. Res., 18:5322 [1990]) andthe long terminal repeats of the Rous sarcoma virus (Gorman et al.,Proc. Natl. Acad. Sci. USA 79:6777 [1982]), and the humancytomegalovirus (Boshart et al., Cell 41:521 [1985]).

[0110] The term “promoter/enhancer,” denotes a segment of DNA whichcontains sequences capable of providing both promoter and enhancerfunctions (for example, the long terminal repeats of retrovirusescontain both promoter and enhancer functions). The enhancer/promoter maybe “endogenous,” or “exogenous,” or “heterologous.” An endogenousenhancer/promoter is one which is naturally linked with a given gene inthe genome. An exogenous (heterologous) enhancer/promoter is one whichis placed in juxtaposition to a gene by means of genetic manipulation(i.e., molecular biological techniques).

[0111] The presence of “splicing signals,” on an expression vector oftenresults in higher levels of expression of the recombinant transcript.Splicing signals mediate the removal of introns from the primary RNAtranscript and consist of a splice donor and acceptor site (Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, New York [1989], pp. 16.7-16.8). A commonly usedsplice donor and acceptor site is the splice junction from the 16S RNAof SV40.

[0112] Efficient expression of recombinant DNA sequences in eukaryoticcells requires signals directing the efficient termination andpolyadenylation of the resulting transcript. Transcription terminationsignals are generally found downstream of the polyadenylation signal andare a few hundred nucleotides in length. The term “poly A site,” or“poly A sequence,” as used herein denotes a DNA sequence which directsboth the termination and polyadenylation of the nascent RNA transcript.Efficient polyadenylation of the recombinant transcript is desirable astranscripts lacking a poly A tail are unstable and are rapidly degraded.The poly A signal utilized in an expression vector may be “heterologous”or “endogenous.” An endogenous poly A signal is one that is foundnaturally at the 3′ end of the coding region of a given gene in thegenome. A heterologous poly A signal is one which is isolated from onegene and placed 3′ of another gene. A commonly used heterologous poly Asignal is the SV40 poly A signal. The SV40 poly A signal is contained ona 237 bp BamHI/BclI restriction fragment and directs both terminationand polyadenylation (Sambrook, supra, at 16.6-16.7). This 237 bpfragment is contained within a 671 bp BamHI/PstI restriction fragment.

[0113] The term “genetically engineered cell line,” refers to a cellline that contains heterologous DNA introduced into the cell line bymeans of molecular biological techniques (i.e., recombinant DNAtechnology).

[0114] The term “stable transfection,” or “stably transfected,” refersto the introduction and integration of foreign DNA into the genome ofthe transfected cell. The term “stable transfectant,” refers to a cellwhich has stably integrated foreign DNA into the genomic DNA.

[0115] The term “stable transfection” (or “stably transfected”), refersto the introduction and integration of foreign DNA into the genome ofthe transfected cell. The term “stable transfectant,” refers to a cellwhich has stably integrated foreign DNA into the genomic DNA.

[0116] The term “selectable marker,” as used herein refers to the use ofa gene which encodes an enzymatic activity that confers resistance to anantibiotic or drug upon the cell in which the selectable marker isexpressed. Selectable markers may be “dominant”; a dominant selectablemarker encodes an enzymatic activity which can be detected in anymammalian cell line. Examples of dominant selectable markers include thebacterial aminoglycoside 3′ phosphotransferase gene (also referred to asthe neo gene) which confers resistance to the drug G418 in mammaliancells, the bacterial hygromycin G phosphotransferase (hyg) gene whichconfers resistance to the antibiotic hygromycin and the bacterialxanthine-guanine phosphoribosyl transferase gene (also referred to asthe gpt gene) which confers the ability to grow in the presence ofmycophenolic acid. Other selectable markers are not dominant in thattheir use must be in conjunction with a cell line that lacks therelevant enzyme activity. Examples of non-dominant selectable markersinclude the thymidine kinase (tk) gene which is used in conjunction withtk⁻ cell lines, the CAD gene which is used in conjunction withCAD-deficient cells and the mammalian hypoxanthine-guaninephosphoribosyl transferase (hprt) gene which is used in conjunction withhprt cell lines. A review of the use of selectable markers in mammaliancell lines is provided in Sambrook et al., supra at pp.16.9-16.15.

[0117] The terms “nucleic acid molecule encoding,” “DNA sequenceencoding,” and “DNA encoding,” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

[0118] The terms “confluent” or “confluency” as used herein in referenceto an adherent cell line define a condition wherein cells throughout aculture are in contact with each other creating what appears to be acontinuous sheet or “monolayer” of cells.

[0119] The terms “cytopathic effect” or “CPE” as used herein describechanges in cellular structure (i.e., a pathologic effect) resulting fromexternal agents such viruses. Common cytopathic effects include celldestruction, syncytia (i.e., fused giant cells) formation, cell roundingvacuole formation, and formation of inclusion bodies. CPE results fromactions of a virus on permissive cells that negatively affect theability of the permissive cellular host to preform its requiredfunctions to remain viable. In in vitro cell culture systems, CPE isevident when cells, as part of a confluent monolayer, show regions ofnon-confluence after contact with a specimen that contains a virus. Theobserved microscopic effect is generally focal in nature and the foci isinitiated by a single virion. However, depending upon viral load in thesample, CPE may be observed throughout the monolayer after a sufficientperiod of incubation. Cells demonstrating viral induced CPE usuallychange morphology to a rounded shape, and over a prolonged period oftime can die and be released form their anchorage points in themonolayer. When many cells reach the point of focal destruction, thearea is called a viral plaque, which appears as a hole in the monolayer.Cytopathic effects are readily discernable and distinguishable by thoseskilled in the art.

[0120] The abbreviation “ONPG,” representso-Nitrophenyl-β-D-Galactopyranoside. ONPG is a substrate for the enzymeβ-galactosidase (β-gal). The reaction between ONPG and β-gal produces ayellow product which can be quantified spectrophotometrically at 405 mn.

[0121] The abbreviation “X-gal,” represents the chemical compound5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, a substrate for theenzyme β-galactosidase. The reaction between X-gal and β-galactosidaseresults in the formation of a blue precipitate which is visuallydiscernable.

[0122] The term “hybriwix,” represents a product of Diagnostic Hybrids,Inc., Athens, Ohio which allows for quantification of certain viral DNAin an infected monolayer of cells by DNA hybridization. “DNAhybridization” is the annealing of two complementary DNA molecules whosebase sequences match according to the rules of base pairing. DNAhybridization is used to identify or quantify an unknown or “target” DNAby hybridization to a known DNA or “probe.” The probe is typicallylabeled with a reporter molecule such as ¹²⁵I, a radioisotope which canbe detected and quantified with a gamma counter.

[0123] The phrase “plaque reduction assay,” or “PRA,” as used hereindescribes a standard method used to determine efficacy of anti-viraldrugs by enumerating a decrease in plaque formation in a cell monolayerexposed to a drug. A “plaque” is a defined area of “CPE.” It is usuallythe result of infection of the cell monolayer with a single infectiousvirus which then replicates and spreads to adjacent cells of themonolayer. A plaque may also be referred to as a “focus of viralinfection.”

[0124] The term “permissive” as used herein describes the sequence ofinteractive events between a virus and its putative host cell. Theprocess begins with viral adsorption to the host cell surface and endswith release of infectious virions. A cell is “permissive” if it readilypermits the spread of virus to other cells. Many methods are availablefor the determination of the permissiveness of a given cell line,including, but not limited to plaque reduction assays, comparisons ofthe production and/or quantitation of viral proteins based on resultsobtained from gel electrophoresis, relative comparisons usinghybridization analysis to analyze DNA or RNA content, etc.

[0125] The term “susceptible,” as used herein describes the extent thata permissive or non-permissive host cell can adsorb and be penetrated bya virus. A cell line may be susceptible without being permissive in thatit can be penetrated but not release virions. A permissive cell linehowever must be susceptible.

[0126] The phrase “seed on,” as used herein describes the act oftransferring an aqueous solution of suspended cells into a vesselcontaining cells adhered to a surface, after which the vessel is storedfor a sufficient period of time to allow the suspended cells or “seeds”to settle out by gravity and attach in a relatively uniform manner tothe adhered cells and become integrated into the final cell monolayer asa mixture. A “mixed cell monolayer,” results from the “seed on” process.

[0127] The phrase “seed in,” as used herein describes the mixing of twoor more aqueous solutions of suspended tissue culture cells, each cellsuspension having different cellular properties, and transfer of suchmixture of cells into a vessel which is stored for a sufficient periodof time to allow the suspended cells to settle out by gravity and attachin a relatively uniform manner such that the distribution of any singlecell type is indicative of the relative ratio of the cells in theoriginal mixture.

[0128] The term “starts,” as used herein refers to the reporter cellswhich represent a primary infection of virus. The virus infects areporter cell (a genetically engineered cell) and induces the expressionof the reporter gene. A reporter cell can be non-permissive (i.e.permissiveness of the reporter cells is not required) and still producestarts.

[0129] EXPERIMENTAL

[0130] The following examples are provided in order to demonstrate andfurther illustrate certain preferred embodiments and aspects of thepresent invention and are not to be construed as limiting the scopethereof.

[0131] In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); μg (micrograms); ng(nanograms); l or L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); xg(times gravity);° C. (degrees Centigrade); FBS (fetal bovine serum); PBS(phosphate buffered saline; HEPES(N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]); HBSS (Hank'sBalanced Salt Solution); MEM (Minimal Essential Medium); EMEM (Eagle'sMinimal Essential Medium); BBL (Becton Dickinson Microbiology Systems,Cockeysville, MD); DIFCO (Difco Laboratories, Detroit, Mich.); U.S.Biochemical (U.S. Biochemical Corp., Cleveland, Ohio); Chemicon(Chemicon, Inc., Temecula, Calif.); Dako (Dako Corporation, Carpinteria,Calif.); Fisher (Fisher Scientific, Pittsburgh, Pa.); Sigma (SigmaChemical Co., St. Louis, Mo.); ATCC (American Type Culture Collection,Rockville, Md.); Bartel's (Bartels, Issaquah, Wash.); and BioWhittaker(BioWhittaker, Walkersville, Md.).

[0132] The cells used during the development of the present inventionand described in the following Examples, were obtained from the ATCC,with the exception being that the BGMK and PRMK cells were obtained fromBioWhittaker, and the MRC-5 cells were obtained from both ATCC andBioWhittaker. The ATCC numbers of the cells are indicated in thefollowing Table. TABLE 2 ATCC Cell Lines Cell Line ATCC NumberBHK/ICP6LacZ-5 CCL-12072 A549 CCL-185 CV-1 CCL-70 HEp-2 CCL-23 hs27 HFF;CRL-1634 Mv1Lu CCL-64 McCoy CCL-1696 NCI-H292 CCL-1848 MRC-5 CCL-171WI-38 CCL-75 Vero CCL-81 MDCK (NBL-2) CCL-34 BHK21 CCL-10 HEL299 CCL-137HeLa CCL-2

EXAMPLE 1 Co-Cultivation of Cells

[0133] In this Example, mixed cell cultures were established in whichsingle, dimorphic cell sheets were produced at confluency.

[0134] In these experiments, all of the cell lines were cultured toconfluency in sterile polystyrene flasks in EMEM (Eagle's MinimalEssential Medium) with 25 mM HEPES, 7% fetal bovine serum (FBS), 2 mML-glutamine, and penicillin/streptomycin (100 Units/100 μtg per ml ofmedium each).

[0135] Cells to be cultured were harvested by first rinsing source cellmonolayers with Hank's Balanced Salt Solution (HBSS) without magnesiumor calcium. Depending upon the cell line, the cells were dissociated byadding trypsin (0.125% in HBSS, without calcium or magnesium) ortrypsin-EDTA (0.25% in 1 mM EDTA in HBSS, without calcium or magnesium),or directly to the cell monolayer, and incubating for approximately 5minutes at ambient temperature. Ten volumes of cell culture medium wasadded to each trypsinized cell suspension and the cells were repeatedlypipetted in order to produce near-single cell suspensions (i.e., withoutcell aggregates). Each trypsinized cell suspension was diluted in anadequate volume of culture medium to produce an optical density of cellsuspension suitable to produce a confluent monolayer of cells within 2-3days of incubation in a 96-well microtiter plate. For single cellmonolayers (i.e., one cell type per well), 0.2 ml of suspension was usedto inoculate each well. For example, the final cell preparations rangedfrom a final optical density at 500 nm of 0.012 OD units/ml for CV-1cells to 0.03 OD units/ml for HEp-2 cells.

[0136] Cell mixture monolayers were produced by co-planting two distinctcell types at an equal volume of each diluted cell suspension (i.e., 0.1ml of each cell type was used to inoculate each well of a 96-wellmicrotiter plate). The cells were allowed to attach to the well surfaceby gravity for 30-60 minutes, and the inoculated microtiter plates wereincubated for up to three days at 36° C. in 5% CO₂ with 95% relativehumidity.

[0137] Periodically during incubation, single and mixed monolayers werechecked for overall viability. The mixed cell culture monolayers werealso checked for the ability of the cell lines to co-exist and developas a single cell sheet (i.e., a single monolayer), with two distinctcell morphologies (i.e., dimorphic cell sheets), at an approximatelyequal density of each cell type. At confluency, the cells were treatedwith a methylene blue staining solution to fix the cells and stain thema light blue in order to provide contrast for visualization using lightmicroscopy.

[0138] Some of the mixed monolayers successfully grew as a mixed cellmonolayer adhered to the well surfaces, exhibiting a smooth, evenlydistributed monolayer. These mixed cultures were designated as“morphologic category 1.” In these cultures, each cell type could beeasily distinguished and appeared to survive well in a mixed monolayer,giving the appearance of a single cell distribution. Mixed monolayerscomposed of HEp-2 and McCoy cells displayed this morphology.

[0139] Some of the mixed monolayers successfully grew as a mixedmonolayer adhered to the well surfaces, but exhibited two distinctmorphologies at confluency. These mixed cultures were designated as“morphologic category 2.” In these cultures, separate, distinct patchesof each cell line co-existed within the monolayer, giving the appearanceof oil mixing with water. Although an understanding of the mechanism isnot necessary in order to use the present invention, it is likely thatthis appearance is most likely the result of contact inhibition betweentwo specific cell types. The relative sizes of the patches was foundprimarily to be a function of how evenly the cells were distributed atcell planting. The more even the cell distribution at planting, thepatches or islands were smaller as the monolayer reached confluency.Examples of monolayers that produced this appearance were mink lungcells co-cultivated with NCI-H292 cells, mink lung cells co-cultivatedby buffalo green monkey kidney (BGMK) cells, and human lung carcinomaA549 cells co-cultivated with NCI-H292 cells.

[0140] However, some cells types could not produce a mixed cellmonolayer, when mixed at relatively equal cell numbers at planting inthe same culture medium. In some of these cultures, only one of the celltypes was found to be viable (i.e., the culture was effectively a singlecell type). Examples of mixed cell cultures that were found to beunsuitable for the production of mixed monolayers include humanembryonic lung fibroblasts (MRC-5 cells) co-cultivated with BGMK cells.In this mixture, the MRC-5 cells become toxic and form aggregates ofdead cells soon after planting. Thus, at confluency, the monolayer onlycontains one functional, viable cell type, the BGMK cells. Thus, thiscell mixture was found to be unsuitable for producing mixed cellmonolayers as the cells failed to form mixed cell monolayers of either asmooth or dimorphic morphologic type.

EXAMPLE 2 Detection of Respiratory Viruses in Mixed Cell Cultures

[0141] In this Example, mixed cell cultures were used to detect variousrespiratory viruses including Influenza A, RSV, adenoviruses,parainfluenza viruses, and Influenza B, present in clinical specimens.The mixed cells used in these experiments were Mv1Lu (mink lung cells)and NCI-H292 (human mucoepidermoid cells).

[0142] Cell Lines

[0143] Confluent T-225 flasks of Mv1Lu and H292 cells were prepared inEMEM with HEPES, 10% FBS, 2 mM L-glutamine, and 50 μg/ml gentamicin. Thecells were harvested by first rinsing them in 30 ml HBSS withoutmagnesium and calcium. The cells were then dissociated from the flask bybrief exposure (i.e., until the cells lifted from the bottom of theflask) to 7 ml trypsin-EDTA solution as described in Example 1. Then, 30ml media was added to the cells to prepare a cell suspensionconcentrate. The optical density of each cell suspension was determinedat 500 nm, using 3 ml of cells. Typically, the OD reading was 0.2/ml forboth the Mv1Lu and H292 cells. In addition to the Mv1Lu and H292 cells,rhesus monkey kidney cells (PRMK), A549 cells, and MDCK cells were usedin the present Example. These additional cell lines were prepared insingle cell cultures as known in the art.

[0144] Mixed Cell Cultures

[0145] When each cell suspension concentrate was determined to be 0.2 ODunits/ml, 5.2 ml of the Mv1Lu, and 8.7 ml H292 cell suspensions wereadded to 86.1 ml of culture medium, in order to provide an acceptableworking ratio of each cell type (i.e., it was a preparation of dilutedmixed cells). This ratio was devised in order to achieve a confluentmonolayer, in which each cell type covered a substantially equivalentsurface area within 1-3 days post-planting of the diluted mixed cells.Prior to dispensing, care was taken to prepare homogenous suspensions ofdiluted mixed cells. The mixed cells were dispensed at 0.75 ml per glassshell vial (i.e., glass vial containing a sterile glass coverslip).After planting, the vials were allowed to sit for 60 minutes at ambienttemperature so that the cells could settle by gravity and produce a moreoptimum cell distribution of each cell type. The mixed cells were thenincubated for 1-3 days at 36° C. in 5% CO₂, at 95% relative humidity.Subsequently, the shell vials were stored at ambient temperature tomaintain each cell type at substantively equivalent surface ratios forup to 10 days from achieving confluency.

[0146] Samples and Processing

[0147] Nasopharyngeal specimens submitted to a diagnostic virologylaboratory were obtained from patients exhibiting influenza-likesymptoms. The specimens were centrifuged to produce a cell pellet fordirect antigen testing, and a specimen supernatant for inoculation ofvarious cell cultures. The cell pellet was resuspended in phosphatebuffer to prepare a cell suspension and 25 μl portions of the cellsuspension were spotted onto a glass slide and dried. Each spot of cellson the slide were then fixed with fixative (e.g., acetone), andincubated for 30 minutes with individual antibody solutions (Bartel's)capable of recognizing various respiratory viruses, including influenzaA and RSV, as well as other respiratory viruses. A second antibodysolution containing fluorescein (FITC) labelled goat anti-mouseantibodies and counterstain (Bartel's) was added to cover each cell spoton the slides, and incubated for an additional 30 minutes at 35-37° C.The counterstain in the FITC-goat anti-mouse antibody solution containsEvans Blue, which stains the cells and appears red under fluorescence.Slides prepared from the nasopharyngeal specimens were observed forpositive (ie., virus-infected), apple green staining fluorescent cells,using epifluorescence at 100-400× magnification.

[0148] In addition, 0.2 ml aliquots of the specimen supernatant wereinoculated onto various cell cultures prepared in shell vials containingglass coverslips. The cell cultures included primary rhesus monkeykidney cells (PRMK; ViroMed or BioWhittaker), Mv1Lu cells (DiagnosticHybrids) HEp-2 cells (Diagnostic Hybrids), MDCK, A549, and H292 cells,as single cell monolayers, as well as mixed cell monolayers of Mv1Lu andH292 cells, produced as described above.

[0149] Each inoculated shell vial was centrifuged for 60 minutes at 700xg, and then incubated for 1-3 days at 36° C., in appropriate culturemedium (e.g., EMEM containing 0.5 to 2% FBS, 2 mM L-glutamine, andpenicillin/streptomycin [100 Units/100 μg per ml of medium each]). Afterincubation, the culture medium was decanted, and the cells were fixed tothe glass coverslip with a solution of acetone and methanol (50:50,v/v). An antibody solution (Chemicon or Bartel's) containing a pool ofmonoclonal antibodies to multiple respiratory viruses, includingInfluenza A and RSV, as well as other respiratory viruses was added tocover each coverslip. The coverslips were then incubated for 30 minutesat 35-37° C. The antibody solution was then removed and the coverslipswere rinsed with PBS. A second antibody solution containing fluorescein(FITC) labelled goat anti-mouse antibodies and counterstain (Bartel's)was added to cover each coverslip, and incubated for an additional 30minutes at 35-37° C. The counterstain in the FITC-goat anti-mouseantibody solution contains Evans Blue, which stains the cells andappears red under fluorescence. Shell vial coverslips prepared from thenasopharyngeal specimens (i.e., inoculated cultures) were observed forpositive (i.e., virus-infected), apple green staining, fluorescentcells, using epifluorescence at 100-400× magnification.

[0150] Results

[0151] Some specimens demonstrated a positive direct antigen reaction onthe cell spot incubated with Influenza A monoclonal antibody. Thesespecimens also demonstrated fluorescent staining on the single cellMv1Lu coverslip and the Mv1Lu/H292 mixed cell coverslip, but no or verylittle fluorescence on the single cell H292 coverslip. The H292 cellsare either not susceptible to this strain of Influenza A, or aresignificantly less susceptible, such that infection is not detectable.Additionally, in some cases (i.e., in specimens with low virus titers),the culture systems were more sensitive than the direct antigendetection method. Also, while the single PRMK cell cultures (i.e., the“gold standard” cells used to detect Influenza A) were positive for thepresence of Influenza A, with many specimens, the numbers of infectedcells and the total of number of positive specimens were lower thanthose identified as positive by the mixed cell monolayers.

[0152] In addition, both the MDCK and PRMK cells missed one low titerspecimen positive for Influenza A by direct antigen testing (IFA), andone other specimen that was also positive for Influenza A by IFA, whilethe Mv1Lu cells detected the virus in all of the samples determined tobe positive based on direct antigen detection (IFA).

[0153] Some specimens demonstrated a positive direct antigen reaction onthe cell spot incubated with RSV monoclonal antibody. These specimensalso demonstrated fluorescent staining on the single cell H292 coverslipand the Mv1Lu/H292 mixed cell coverslip, but no or very littlefluorescence on the single cell MV1Lu coverslip. H292 cells aresusceptible to RSV infection, while Mv1Lu cells are not susceptible (orare significantly less susceptible, such that infection is notdetectable). In addition to the mixed cell cultures, HEp-2 cells (i.e.,the “gold standard” cells used to detect RSV) were also observed for thepresence of RSV; the performance of HEp-2 cells was generally lesssensitive than that of the Mv1Lu and H292 mixed cell monolayers, or theH292 single cell monolayers. These results with Influenza A tested inmink lung cells was very surprising, as the detection of Influenza Ausing these cells has previously not been described.

[0154] Adenoviruses identified from five clinical specimens based ondirect antigen testing (IFA) were detected in the H292 and cell culturemixes, while the PRMK cells missed two of the low titer specimens (i.e.,there were two false negatives). Thus, H292 and the mixed cultures weremore sensitive than PRMK for detection of adenoviruses. While the A549cells may provide slightly more positive cells, the 292 cells, mixedcell cultures, and A549 cells detected an equal number of positivespecimens.

[0155] Parainfluenza viruses were also detected in the H292 and mixedcell cultures, while the PRMK cells missed one low titer specimen.

[0156] These results clearly show that the mixed cell cultures wereequal in sensitivity to the single cell (H292 and Mv1Lu) cultures. Thus,the mixed cells provide savings in material, time, space, and labor,while providing the same level of sensitivity in the detection ofrespiratory viruses as single cell cultures presently commonly used indiagnostic virology laboratories.

[0157] Influenza B Specimens

[0158] In addition to the samples discussed above, various dilutions ofmultiple Influenza B strains obtained from the ATCC were tested in MDCK,Mv1Lu, and PRMK cells. The following table provides the results of theseexperiments. In this Table, “MD” refers to the “Maryland” strain, “HK”refers to the “Hong Kong” strain, “TW” refers to the “Taiwan” strain,and “MA” refers to the “Massachusetts” strain. TABLE 3 Comparison ofInfluenza B Virus Detection From Prototype Viruses by MDCK, ML, and PRMKCells Influenza B Virus Virus Cell Line Strain Dilution MDCK Mv1Lu PRMKMD 10⁻⁴ + + + 10⁻⁵ + + + 10⁻⁶ − + − HK 10⁻⁴ + + + 10⁻⁵ + + − 10⁻⁶ − − −TW 10⁻⁴ + + + 10⁻⁵ + + + 10⁻⁶ − − − MA 10⁻⁴ + + + 10⁻⁵ + + + 10⁻⁶ + + +

EXAMPLE 3

[0159] Detection of CMV in Mixed Cell Cultures

[0160] In this Example, mixed cell cultures of Mv1Lu and NCI-H292 cellswere used to detect the presence of human cytomegalovirus (HCMV).

[0161] The Towne strain of HCMV (ATCC #VR977) was amplified in MRC-5cells to a titer of greater than 10⁶/ml, and frozen at −85° C. in EMEMcontaining 10% FBS. Serial dilutions of HCMV were prepared andinoculated into single monolayers of mink lung (Mv1Lu) cells, MRC-5cells, and mixed cell monolayers of Mv1Lu and H292 cells. Each infectedcell culture system was centrifuged for 60 minutes at 700 xg, and thenincubated for 24 hours at 36° C. in 5% CO₂, in appropriate culturemedium (e.g., EMEM containing 10% FBS). The culture medium was removedand the cells were fixed to the glass coverslip using a solution of 80%acetone in water. A sufficient amount of HCMV antibody solution(Chemicon) was added to cover each coverslip and incubated for 30minutes at 35-37° C. The antibody solution was removed, and thecoverslip was rinsed with PBS. A second antibody solution consisting ofFITC-labelled goat anti-mouse antiserum was added to cover eachcoverslip and incubated an additional 30 minutes at 35-37° C. Thespecimens were then observed under epifluorescence at 100-400×magnification for positive (i.e., CMV-infected), nuclear staining,fluorescent cells.

[0162] As described in previous Examples, the counterstain in theFITC-labelled goat anti-mouse antibody solution contains Evans Blue,which stains the cells and appears red, when excited by fluorescentlight. Fluorescent, apple green nuclear stain was observed in the Mv1Lusingle cell monolayer and in the mixed cell monolayers, but not in theH292 cells, as the Mv1Lu cells are susceptible to HCMV infection, whileH292 cells are not (or the H292 cells are significantly less sensitive).The MRC-5 cells (i.e., the “gold standard” cells for detection of HCMV)performed about the same as the mixed cell monolayer, as these cultureshad a similar number of infected cells as the cells in the mixedmonolayer.

EXAMPLE 4 Detection of Enteroviruses in Mixed Cell Cultures

[0163] In this Example, mixed cell cultures were used to detect theenteroviruses, Coxsackie B virus and Echovirus. In these experiments, amixed cell monolayer of BGMK and NCI-H292 cells were used.

[0164] Confluent T-225 flasks of BGMK and H292 cells were prepared inEMEM with 25 mM HEPES, 10% FBS, 2 mM L-glutamine, and 50 μg/mlgentamicin. The cells were harvested by first rinsing in 30 ml HBSSwithout magnesium and calcium, and were then dissociated from the flasksby a brief treatment of 7 ml trypsin-EDTA solution (as described inExample 1). Then, an additional 30 ml of culture medium (EMEM withHEPES, 10% FBS, 2 mM L-glutamine, and 50 μg/ml gentamicin) was added tothe suspension to produce a cell suspension concentrate. The opticaldensity at 500 nm was determined for each suspension, using 3 ml ofcells. Typically, the OD reading was 0.2/ml for both the BGMK and H292cell suspensions.

[0165] Next, 3 ml of BGMK cell suspension and 8 ml of H292 cellsuspension (both suspensions were at 0.2 OD units/ml) were then added to29 ml of the culture medium (25 mM HEPES, 10% FBS, 2 mM L-glutamine, and50 μg/ml gentamicin) to provide an acceptable working ratio of each celltype in a diluted mixed cell suspension. This ratio was intended toachieve a confluent monolayer consisting of each cell type coveringsubstantially equivalent surface area within 1-3 days post-planting ofthe diluted mixed cells. Care was exercised to prepare a homogenoussuspension of diluted mixed cells prior to dispensing 0.75 ml to each of100 glass shell vials, each of which contained a sterile glasscoverslip. The vials were allowed to sit for 60 minutes post-planting atambient temperature to allow the cells to settle by gravity and producea more optimum cell distribution. The vials were then were moved to anincubator for incubation at 36° C. for 1-3 days in 5% CO₂, at 95%relative humidity.

[0166] Stock virus suspensions and clinical specimens shown to containCoxsackie B virus or echovirus were used to infect BGMK/H292 cellmixtures, as well as single cell monolayers of BGMK, H292, MRC-5, andPRMK cells. For clinical samples, throat swab, nasopharyngeal swab,sputum, stool, and rectal swabs were collected from patients, placed inviral transport medium, and filtered through 0.45 μm filter to removepossible bacterial and fungal contaminants prior to inoculation of cellcultures. Cerebrospinal fluid (CSF) collected from patients was placedin viral transport medium, and used directly for inoculation of cells.For inoculation of shell vials, the media present in the vials wereremoved and fresh media added. Then, 0.2 ml of specimen was inoculatedinto each vial. The inoculated vials were centrifuged at 700 xg for45-60 minutes at room temperature. Subsequently, the vials wereincubated at 37° C. for 1-3 days, and viral presence was detected usingimmunofluorescent staining.

[0167] For staining, the medium was removed from each vial and the cellswere fixed on the coverslip with acetone. The coverslip was removed fromeach vial, and stained with 25 μl primary antibody (mouse monoclonalantibody directed against enteroviruses [Dako]), for 30 minutes at 37°C. After washing with PBS, 25 μl of the FITC-conjugated anti-mouse Ig(Dako) was used as a secondary antibody for staining, and incubated at37° C. for 30 minutes. After another wash, the coverslips were mountedon slides and observed under fluorescence. The presence of one or morespecific fluorescent-stained cells on the coverslip was considered to bea positive. As described in previous Examples, the counterstain in theFITC-labelled goat anti-mouse antibody solution contains Evans Blue,which stains the cells, and appears red upon exposure to fluorescentlight. For Coxsackie B virus detection, fluorescent, apple green stainwas observed in many of the BGMK cells in the BGMK single cell monolayerand in the mixed cell monolayers primarily in the BGMK cells, but not inas many H292 cells, as BGMK cells are more susceptible to Coxsackie Bvirus infection. For some types of Coxsackie B virus isolates, H292cells are not as susceptible (or the H292 cells are significantly lesssusceptible). The “gold standard” cell line (i.e., PRMK cells) did notexhibit the same number of infected cells as the mixed cell monolayers.

[0168] For detection of echovirus, fluorescent, apple green stain wasobserved in many H292 cells in the H292 single cell monolayer and in themixed cell monolayers, primarily in the H292 cells, but not in as manyBGMK cells, as H292 cells are more susceptible to echovirus infection,while BGMK cells are not as susceptible (or the BGMK cells aresignificantly less sensitive). The “gold standard” line (i.e., MRC-5cells) performed, but did not appear to have as many infected cells asthe mixed cell monolayers. In the case of the BGMK/H292 mixed cellmonolayers infected with high titer samples of enteroviruses,cell-specific virus mediated cytopathic effect (CPE) was evident (i.e.,the CPE was observed in BGMK cells when Coxsackie B virus was present athigh titer, and CPE was observed in H292 cells when echovirus waspresent at high titer).

EXAMPLE 5 Detection of Herpes Simplex Virus and HCMV in Mixed CellCultures

[0169] In this Example, mixed cell cultures are used to detect herpessimplex virus (HSV) and HCMV, using a mixed cell monolayer ofgenetically engineered baby hamster kidney (BHK) cells (e.g., ATCC#CCL-12072) and Mv1Lu cells.

[0170] The BHK and Mv1Lu cells are grown in flasks, trypsinized, andmixed as described in previous Examples, such that a suitable dilutionof mixed cells is produced. These mixed cell dilutions are then used toinoculate sterile glass shell vials containing coverslips, as describedabove. The cells are then centrifuged and inoculated with virus orclinical samples, incubated, and fixed, as described above.

[0171] HCMV is detected in the Mv1Lu cells, using antibody as describedin Example 3 above, and HSV (HSV-1 and HSV-2) are identified using aβ-galactosidase staining kit (i.e., detecting the reporter gene inducedby the virus infecting the genetically engineered BHK cells).

EXAMPLE 6 Detection of Respiratory Viruses in Mixed Cell Cultures

[0172] In this Example, mixed cell cultures are used to detect a panelof respiratory viruses. In these experiments, three cell types arecombined so as to produce a mixed cell culture that is capable ofdetecting at least three viruses.

[0173] First, A549, H292, and mink lung (e.g., Mv1Lu) cells cells aregrown in flasks, trypsinized, and mixed as described in previousExamples, such that a suitable dilution of mixed cells is produced. Inpreferred embodiments, the cells are diluted such that the mixed cellsin culture will be in approximately the same proportions (i.e., 1:1:1).These mixed cell dilutions are then used to inoculate sterile glassshell vials containing coverslips, as described above. The cells arethen centrifuged and inoculated with virus or clinical samples,incubated, and fixed, as described above.

[0174] The viruses capable of infecting these cells are detected andidentified using the methods described in Example 2 above. In thesemixed cell cultures, the 292 cells are used to detect the presence ofparainfluenza viruses and RSV, while the A549 cells are used to detectthe presence of adenoviruses, and the mink lung cells are used to detectthe presence of influenza viruses (e.g., Influenza A and B).

EXAMPLE 7 Detection of HSV and Chlamydia in Mixed Cell Cultures

[0175] In this Example, mixed cell cultures are provided which allow thedetection of two organisms commonly associated with sexually transmitteddiseases. In these experiments, mink lung cells (e.g., Mv1Lu) useful forthe detection of HSV are mixed with McCoy cells useful for the detectionof C. trachomatis.

[0176] First, McCoy cells and mink lung (e.g., Mv1Lu) cells cells aregrown in flasks, trypsinized, and mixed as described in previousExamples, such that a suitable dilution of mixed cells is produced. Inpreferred embodiments, the cells are diluted such that the mixed cellsin culture will be in approximately the same proportions. These mixedcell dilutions are then used to inoculate sterile glass shell vialscontaining coverslips, as described above. The cells are thencentrifuged and inoculated with samples (e.g., clinical samples),incubated, and fixed, as described above.

[0177] The organisms capable of infecting these cells (e.g., HSV infectsthe mink lung cells, while C. trachomatis infects the McCoy cells) aredetected and identified using the methods described in Example 2 above.As with the other mixed cell culture systems, the presence of virusand/or C. trachomatis may be detected by other methods, such as theobservation of CPE, animal inoculation, etc. Thus, it is not intendedthat the mixed cell culture assay systems of this Example or any of thepreceding examples be limited to any particular method of microorganismdetection, identification, and/or quantitation.

EXAMPLE 8 Detection of Enteroviruses In Mixed Cell Cultures of HumanEmbryonal Rhabdomyosarcoma (RD) Cells and H292 Cells, and In Mixed CellCultures of BGMK and A549 Cells

[0178] In this Example, two different mixed cell cultures [Mix Acontaining RD cells (ATCC #CCL 136) and H292 cells; Mix B containingBGMK cells and A549 cells] were used to detect eighty (80) differentisolates of enteroviruses. The mixed cell cultures were preparedessentially as described in Example 1, supra. The eighty isolates werestored (frozen) clinical isolates which had been harvested on MRC5and/or PMK from cerebral spinal fluid, throat, and feces. The resultsare shown in Table 4. TABLE 4 Percentage of Isolates with Growth in MixA and Mix B Virus (Number of Growth Growth in Either Isolates Tested) inMix A Growth in Mix B Mix A or Mix B Coxsackie (36) 64% 75% 100% Echo(37) 95% 51% 100% Polio (7) 100%  100%  100% All Enteroviruses (80) 81%66% 100%

[0179] Of the Echo viruses, 95% grew on Mix A; 51% grew on Mix B. Allthe Polio viruses grew on both Mix A and B.

[0180] These results demonstrate that each of Mix A and Mix B wassuccessful in detecting the vast majority (81% and 60%, respectively) ofall enteroviruses tested. The data also demonstrate that the combinationof Mix A and Mix B detected 100% of all the clinical isolates ofenteroviruses tested. Thus, the combination of Mix A and Mix B makes itpossible to detect all enteroviruses from clinical sources such ascerebral spinal fluid, throat, and feces.

EXAMPLE 9 Detection of Enteroviruses In Mixed Cell Cultures ContainingHuman Colon Adenocarcinoma (Caco-2) Cells

[0181] In this Example, mixed cell cultures containing human colonadenocarcinoma (Caco-2) cells (ATCC #HTB 37) are used to detect andidentify enteric viruses, including coxsakieviruses, echoviruses,polioviruses, rotaviruses, astroviruses, and adenoviruses. Rotaviruses,astroviruses, and adenoviruses are responsible for 80% of viralgastroenteritis occurring in young children [Pinto et al. (1994) J.Medical Virol. 44:310-315].

[0182] Mixed Cell Cultures

[0183] Mixed cell cultures containing human colon adenocarcinoma(Caco-2) cells (ATCC #HTB 37) in combination with one, two and threeother cell types selected from RD cells, H292 cells, BGMK cells, andA549 cells (Table 5) are used to detect eighty (80) different isolatesof enteroviruses, which are prepared as described above in Example 8.The mixed cell cultures are also used to detect polio virus 1,coxsakievirus A24, enterovirus 70, reovirus 3, human rotavirus,Adenovirus 5, Adenovirus 40, and Adenovirus 41, astrovirus 1, and HAVstrain HM175. These viruses have been previously described by Pinto etal. (1994), supra. The mixed cell cultures described in the followingTable 5 are prepared essentially as described in Example 1, supra, andare compared to single cell type cultures which contain Caco-2, RD,H292, BGMK, or A549 cells. TABLE 5 Mixed Cultures Containing Caco-2Cells Sample Number Cell Lines 1 Caco-2, RD 2 Caco-2, H292 3 Caco-2,BGMK 4 Caco-2, A549 5 Caco-2, RD, H292 6 Caco-2, RD, BGMK 7 Caco-2, RD,A549 8 Caco-2, RD, H292, BGMK 9 Caco-2, RD, H292, A549 10 Caco-2, RD,BGMK, A549 11 Caco-2, RD, H292, BGMK, A549

[0184] Periodically during incubation, single and mixed monolayers arechecked for overall viability and ability of the cell lines to co-existand develop as a single cell sheet (i.e., a single monolayer), with twodistinct cell morphologies (i.e., dimorphic cell sheets), at anapproximately equal density of each cell type. Successful mixed culturesare those in which mixed cell monolayers adhere to the well surfaces,exhibiting a smooth, evenly distributed monolayer in which each celltype can be easily distinguished (i.e., “morphologic category 1”).Alternatively, successful mixed cultures are those in which mixedmonolayers adhere to the well surfaces, but exhibit two distinctmorphologies at confluency (i.e., “morphologic category 2”).

[0185] Samples and Processing

[0186] Enteric viruses are detected by identification of virus-inducedcytopathic effect (CPE), and/or by detection of expression of at leastone early virus-specific nucleic acid before CPE is evident in theinfected cultures. The presence of enteric viruses is assayed bydot-blot hybridization before and after infection of mixed cell cultureswhich contain Caco-2 cells as previously described [Pinto et al. (1994)supra]. Briefly, virus RNA and DNA is extracted from stool suspensionsand at 48 hours post-infection (p.i.) of mixed cell cultures. A probecorresponding to the 2,300 bp of gene 4 of rotavirus Wa is used todetect human rotavirus; a probe corresponding to 1 kb from the 3′ end ofhuman astrovirus type 1 is used to detect human astroviruses; and aprobe corresponding to the 1,800 bp PstI fragment H of Adenovirus 40,and a probe corresponding to the 6,500 bp PstI fragment B of adenovirus41 is used to detect Adenovirus 40 and Adenovirus 41, respectively[Pinto et al. (1994) supra]. All cDNA probes are labelled withdigoxigenin-11-UTP (Boehringer Mannheim) following the manufacturer'sinstructions. A dot-blot hybridization assay is carried out with thedifferent cDNA probes following standard procedures.

[0187] Results

[0188] The detection of CPE and/or virus nucleic acids using dot-blothybridization in mixed cell cultures containing Caco-2 cellsdemonstrates that these cells are susceptible to the enteric viruses(i.e., coxsakieviruses, echoviruses, polioviruses, rotaviruses,astroviruses, and adenoviruses) with which the mixed cell cultures areinoculated.

[0189] From the above, it is clear that the present invention providesmany advantages over presently used methods in diagnostic microbiology.

[0190] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in diagnostic microbiology and virology, cell culture,and/or related fields are intended to be within the scope of thefollowing claims.

What is claimed is:
 1. A method for the detection of enterovirus in asample, comprising: a) providing: i) a sample suspected of containingenterovirus; and ii) a mixed cell culture comprising at least two celltypes, wherein said cell types comprise RD cells and humanmucoepidermoid cells; b) inoculating said mixed cell culture with atleast a portion of said sample to produce an inoculated culture; and c)observing said inoculated culture for the presence of said enterovirus.2. The method of claim 1, wherein said human mucoepidernoid cell isH292.
 3. The method of claim 1, wherein said enterovirus is selectedfrom Coxsackie viruses, echoviruses, and polioviruses.
 4. A method forthe detection of enterovirus in a sample, comprising: a) providing: i) asample suspected of containing enterovirus; and ii) a mixed cell culturecomprising at least two cell types, wherein one of said cell typescomprises Caco-2 cells; b) inoculating said mixed cell culture with atleast a portion of said sample to produce an inoculated culture; and c)observing said inoculated culture for the presence of said enterovirus.5. The method of claim 4, wherein said cell type other than said Caco-2cells is selected from RD cells, H292 cells, BGMK cells, and A549 cells.6. The method of claim 4, wherein said enterovirus is selected fromCoxsackie viruses, echoviruses, polioviruses, rotaviruses, astroviruses,and adenoviruses.
 7. A method for the detection and identification ofparainfluenza virus in a sample, comprising the steps of: a) providing:i) a sample suspected of containing parainfluenza virus; and ii) a mixedcell culture comprising at least two cell types, wherein one of saidcell types comprises mink lung cells; b) inoculating said mixed cellculture with at least a portion of said sample to produce an inoculatedculture; and c) observing said inoculated culture for the presence ofsaid parainfluenza virus.
 8. The method of claim 7, wherein said minklung cells are Mv1Lu cells.
 9. The method of claim 7, wherein saidparainfluenza virus is selected from type 1, type 2 and type 3.